Optimization of immunomodulatory properties of genetic vaccines

ABSTRACT

This invention provides methods for obtaining molecules that can modulate an immune response, and immunomodulatory molecules obtained using the methods. The molecules find use, for example, in the tailoring of an immune response induced by a genetic vaccine for a desired purpose.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims benefit of U.S. Provisional Application No.60/074,294, filed Feb. 11, 1998, which application is incorporatedherein by reference for all purposes.

STATEMENT REGARDING GOVERNMENT SUPPORT

This invention was made with Government support under Grant No.N65236-98-1-5401, awarded by the Defense Advanced Projects Agency. TheGovernment has certain rights in this invention.

BACKGROUND OF THE INVENTION

1. Field of the Invention

This invention pertains to the field of modulation of immune responsessuch as those induced by genetic vaccines.

2. Background

Antigen processing and presentation is only one factor which determinesthe effectiveness of vaccination, whether performed with geneticvaccines or more classical methods. Other molecules involved indetermining vaccine effectiveness include cytokines (interleukins,interferons, chemokines, hematopoietic growth factors, tumor necrosisfactors and transforming growth factors), which are small molecularweight proteins that regulate maturation, activation, proliferation anddifferentiation of the cells of the immune system. Characteristicfeatures of cytokines are pleiotropy and redundancy; that is, onecytokine often has several functions and a given function is oftenmediated by more than one cytokine. In addition, several cytokines haveadditive or synergistic effects with other cytokines, and a number ofcytokines also share receptor components.

Due to the complexity of the cytokine networks, studies on thephysiological significance of a given cytokine have been difficult,although recent studies using cytokine gene-deficient mice havesignificantly improved our understanding on the functions of cytokinesin vivo. In addition to soluble proteins, several membrane-boundcostimulatory molecules play a fundamental role in the regulation ofimmune responses. These molecules include CD40, CD40 ligand, CD27, CD80,CD86 and CD150 (SLAM), and they are typically expressed on lymphoidcells after activation via antigen recognition or through cell-cellinteractions.

T helper (T_(H)) cells, key regulators of the immune system, are capableof producing a large number of different cytokines, and based on theircytokine synthesis pattern T_(H) cells are divided into two subsets(Paul and Seder (1994) Cell 76: 241-251). T_(H)1 cells produce highlevels of IL-2 and IFN-γ and no or minimal levels of IL-4, IL-5 andIL-13. In contrast, T_(H)2 cells produce high levels of IL-4, IL-5 andIL-13, and IL-2 and IFN-γ production is minimal or absent. T_(H)1 cellsactivate macrophages, dendritic cells and augment the cytolytic activityof CD8⁺ cytotoxic T lymphocytes and NK cells (Id.), whereas T_(H)2 cellsprovide efficient help for B cells and they also mediate allergicresponses due to the capacity of T_(H)2 cells to induce IgE isotypeswitching and differentiation of B cells into IgE secreting cell (DeVries and Punnonen (1996) In Cytokine regulation of humoral immunity:basic and clinical aspects. Eds. Snapper, C. M., John Wiley & Sons,Ltd., West Sussex, UK, p. 195-215). The exact mechanisms that regulatethe differentiation of T helper cells are not fully understood, butcytokines are believed to play a major role. IL-4 has been shown todirect T_(H)2 differentiation, whereas IL-12 induces development ofT_(H)1 cells (Paul and Seder, supra.). In addition, it has beensuggested that membrane bound costimulatory molecules, such as CD80,CD86 and CD150, can direct T_(H)1 and/or T_(H)2 development, and thesame molecules that regulate T_(H) cell differentiation also affectactivation, proliferation and differentiation of B cells intoIg-secreting plasma cells (Cocks et al. (1995) Nature 376: 260-263;Lenschow et al. (1996) Immunity 5: 285-293; Punnonen et al. (1993) Proc.Nat'l. Acad. Sci. USA. 90: 3730-3734; Punnonen et al. (1997) J. Exp.Med. 185: 993-1004).

Studies in both man and mice have demonstrated that the cytokinesynthesis profile of T helper (T_(H)) cells plays a crucial role indetermining the outcome of several viral, bacterial and parasiticinfections. High frequency of T_(H)1 cells generally protects fromlethal infections, whereas dominant T_(H)2 phenotype often results indisseminated, chronic infections. For example, T_(H)1 phenotype isobserved in tuberculoid (resistant) form of leprosy and T_(H)2 phenotypein lepromatous, multibacillary (susceptible) lesions (Yamamura et al.(1991) Science 254: 277-279). Similarly, late-stage HIV patients haveT_(H)2-like cytokine synthesis profiles, and T_(H)1 phenotype has beenproposed to protect from AIDS (Maggi et al (1994) J. Exp. Med. 180:489-495). Furthermore, the survival from meningococcal septicemia isgenetically determined based on the capacity of peripheral bloodleukocytes to produce TNF-α and IL-10. Individuals from families withhigh production of IL-10 have increased risk of fatal meningococcaldisease, whereas members of families with high TNF-α production weremore likely to survive the infection (Westendorp et al. (1997) Lancet349: 170-173).

Cytokine treatments can dramatically influence T_(H)1/T_(H)2 celldifferentiation and macrophage activation, and thereby the outcome ofinfectious diseases. For example, BALB/c mice infected with Leishmaniamajor generally develop a disseminated fatal disease with a T_(H)2phenotype, but when treated with anti-IL-4 mAbs or IL-12, the frequencyof T_(H)1 cells in the mice increases and they are able to counteractthe pathogen invasion (Chatelain et al. (1992) J. Immunol. 148:1182-1187). Similarly, IFN-γ protects mice from lethal Herpes SimplexVirus (HSV) infection, and MCP-1 prevents lethal infections byPseudomonas aeruginosa or Salmonella typhimurium. In addition, cytokinetreatments, such as recombinant IL-2, have shown beneficial effects inhuman common variable immunodeficiency (Cunningham-Rundles et al. (1994)N. Engl. J. Med. 331: 918-921).

The administration of cytokines and other molecules to modulate immuneresponses in a manner most appropriate for treating a particular diseasecan provide a significant tool for the treatment of disease. However,presently available immunomodulator treatments can have severaldisadvantages, such as insufficient specific activity, induction ofimmune responses against, the immunomodulator that is administered, andother potential problems. Thus, a need exists for immunomodulators thatexhibit improved properties relative to those currently available. Thepresent invention fulfills this and other needs.

SUMMARY OF THE INVENTION

The present invention provides methods of obtaining a polynucleotidethat has a modulatory effect on an immune response that is induced by agenetic vaccine either directly (i.e., as an immunomodulatorypolynucleotide) or indirectly (i.e., upon translation of thepolynucleotide to create an immunomodulatory polypeptide. The methods ofthe invention involve: creating a library of recombinantpolynucleotides; and screening the library to identify at least oneoptimized recombinant polynucleotide that exhibits, either by itself orthrough the encoded polypeptide, an enhanced ability to modulate animmune response than a form of the nucleic acid from which the librarywas created. Examples include, for example, CpG-rich polynucleotidesequences, polynucleotide sequences that encode a costimulator (e.g.,B7-1, B7-2, CD1, CD40, CD154 (ligand for CD40), CD150 (SLAM), or acytokine. The screening step used in these methods can include, forexample, introducing genetic vaccine vectors which comprise the libraryof recombinant nucleic acids into a cell, and identifying cells whichexhibit an increased ability to modulate an immune response of interestor increased ability to express an immunomodulatory molecule. Forexample, a library of recombinant cytokine-encoding nucleic acids can bescreened by testing the ability of cytokines encoded by the nucleicacids to activate cells which contain a receptor for the cytokine. Thereceptor for the cytokine can be native to the cell, or can be expressedfrom a heterologous nucleic acid that encodes the cytokine receptor. Forexample, the optimized costimulators can be tested to identify those forwhich the cells or culture medium are capable of inducing apredominantly T_(H)2 immmune response, or a predominantly T_(H)1 immuneresponse.

In some embodiments, the polynucleotide that has a modulatory effect onan immune response is obtained by: (1) recombining at least first andsecond forms of a nucleic acid that is, or encodes a molecule that is,involved in modulating an immune response, wherein the first and secondforms differ from each other in two or more nucleotides, to produce alibrary of recombinant polynucleotides; and (2) screening the library toidentify at least one optimized recombinant polynucleotide thatexhibits, either by itself or through the encoded polypeptide, anenhanced ability to modulate an immune response than a form of thenucleic acid from which the library was created. If additionaloptimization is desired, the method can further involve: (3) recombiningat least one optimized recombinant polynucleotide with a further form ofthe nucleic acid, which is the same or different from the first andsecond forms, to produce a further library of recombinantpolynucleotides; (4) screening the further library to identify at leastone further optimized recombinant polynucleotide that exhibits anenhanced ability to modulate an immune response than a form of thenucleic acid from which the library was created; and (5) repeating (3)and (4), as necessary, until the further optimized recombinantpolynucleotide exhibits an further enhanced ability to modulate animmune response than a form of the nucleic acid from which the librarywas created.

In some embodiments of the invention, the library of recombinantpolynucleotides is screened by: expressing the recombinantpolynucleotides so that the encoded peptides or polypeptides areproduced as fusions with a protein displayed on the surface of areplicable genetic package; contacting the replicable genetic packageswith a plurality of cells that display the receptor; and identifyingcells that exhibit a modulation of an immune response mediated by thereceptor.

The invention also provides methods for obtaining a polynucleotide thatencodes an accessory molecule that improves the transport orpresentation of antigens by a cell. These methods involve creating alibrary of recombinant polynucleotides by subjecting to recombinationnucleic acids that encode all or part of the accessory molecule; andscreening the library to identify an optimized recombinantpolynucleotide that encodes a recombinant accessory molecule thatconfers upon a cell an increased or decreased ability to transport orpresent an antigen on a surface of the cell compared to an accessorymolecule encoded by the non-recombinant nucleic acids. In someembodiments, the screening step involves: introducing the library ofrecombinant polynucleotides into a genetic vaccine vector that encodesan antigen to form a library of vectors; introducing the library ofvectors into mammalian cells; and identifying mammalian cells thatexhibit increased or decreased immunogenicity to the antigen.

In some embodiments of the invention, the cytokine that is optimized isinterleukin-12 and the screening is performed by growing mammalian cellswhich contain the genetic vaccine vector in a culture medium, anddetecting whether T cell proliferation or T cell differentiation isinduced by contact with the culture medium. In another embodiment, thecytokine is interferon-α and the screening is performed by expressingthe recombinant vector module as a fusion protein which is displayed onthe surface of a bacteriophage to form a phage display library, andidentifying phage library members which are capable of inhibitingproliferation of a B cell line. Another embodiment utilizes B7-1 (CD80)or B7-2 (CD86) as the costimulator and the cell or culture medium istested for ability to modulate an immune response.

The invention provides methods of using DNA shuffling to obtainoptimized recombinant vector modules that encode cytokines and othercostimulators that exhibit reduced immunogenicity compared to acorresponding polypeptide encoded by a non-optimized vector module. Thereduced immunogenicity can be detected by introducing a cytokine orcostimulator encoded by the recombinant vector module into a mammal anddetermining whether an immune response is induced against the cytokine.

The invention also provides methods of obtaining optimizedimmunomodulatory sequences that encode a cytokine antagonist. Forexample, suitable cytokine agonists include a soluble cytokine receptorand a transmembrane cytokine receptor having a defective signalsequence. Examples include ΔIL-10R and ΔIL-4R, and the like.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows an example of a cytotoxic T-cell inducing sequence (CTIS)obtained from HBsAg polypeptide (PreS2 plus S regions).

FIG. 2 shows a CTIS having heterologous epitopes attached to thecytoplasmic portion.

FIG. 3 shows the derivation of immunogenic agonistic sequences (IAS) asdescribed in Example 3. Specific killing (percent) is shown for aneffector: target (E:T) ratio of five.

FIG. 4 shows a method for preparing immunogenic agonist sequences (IAS).Wild-type (WT) and mutated forms of nucleic acids encoding a polypeptideof interest are assembled and subjected to DNA shuffling to obtain anucleic acid encoding a poly-epitope region that contains potentialagonist sequences.

FIG. 5 shows a scheme for improving immunostimulatory sequences by DNAshuffling.

FIG. 6 is a diagram of a procedure by which recombinant libraries ofhuman IL-12 genes can be screened to identify shuffled IL-12 genes thatencode recombinant IL-12 having increased ability to induce T cellproliferation.

FIG. 7 shows the results of a high-throughput functional assay forvectors that encode variants of IL-12 obtained using the methods of theinvention.

FIG. 8 shows the induction of T cell proliferation upon transfection ofthe T cells by individual vectors that encode IL-12 variants.

FIG. 9 shows results of an experiment which demonstrates that a shuffledIL-12 chimera obtained using the methods of the invention exhibitsimproved ability to activate human T cells.

FIG. 10 shows a model of how T cell activation or anergy can be inducedby genetic vaccine vectors that encode different B7-1 (CD80) and/or B7-2(CD86) variants.

FIG. 11 shows a method for using DNA shuffling to obtain CD80/CD86variants that have improved capacity to induce T cell activation oranergy.

FIG. 12 shows results obtained in a screening assay for altered functionof B7.

FIG. 13 provides experimental results which demonstrate that shuffled B7chimeras provide potent T cell activation.

FIG. 14 presents an alignment of the nucleotide sequences for human andmouse IL-10 receptor sequences.

FIG. 15 shows an alignment of the nucleotide sequences of B7-1 (CD80)genes from human, rhesus monkey, and rabbit.

DETAILED DESCRIPTION

Definitions

The term “cytokine” includes, for example, interleukins, interferons,chemokines, hematopoietic growth factors, tumor necrosis factors andtransforming growth factors. In general these are small molecular weightproteins that regulate maturation, activation, proliferation anddifferentiation of the cells of the immune system.

The term “screening” describes, in general, a process that identifiesoptimal immunomodulatory molecules. Several properties of the respectivemolecules can be used in selection and screening including, for example,ability to induce a desired immune response in a test system. Selectionis a form of screening in which identification and physical separationare achieved simultaneously by expression of a selection marker, which,in some genetic circumstances, allows cells expressing the marker tosurvive while other cells die (or vice versa). Screening markersinclude, for example, luciferase, beta-galactosidase and greenfluorescent protein. Selection markers include drug and toxin resistancegenes, and the like. Because of limitations in studying primary immuneresponses in vitro, in vivo studies are particularly useful screeningmethods. In these studies, the genetic vaccines that includeimmunomodulatory molecules are first introduced to test animals, and theimmune responses are subsequently studied by analyzing protective immuneresponses or by studying the quality or strength of the induced immuneresponse using lymphoid cells derived from the immunized animal.Although spontaneous selection can and does occur in the course ofnatural evolution, in the present methods selection is performed by man.

A “exogenous DNA segment”, “heterologous sequence” or a “heterologousnucleic-acid”, as used herein, is one that originates from a sourceforeign to the particular host cell, or, if from the same source, ismodified from its original form. Thus, a heterologous gene in a hostcell includes a gene that is endogenous to the particular host cell, buthas been modified. Modification of a heterologous sequence in theapplications described herein typically occurs through the use of DNAshuffling. Thus, the terms refer to a DNA segment which is foreign orheterologous to the cell, or homologous to the cell but in a positionwithin the host cell nucleic acid in which the element is not ordinarilyfound. Exogenous DNA segments are expressed to yield exogenouspolypeptides.

The term “gene” is used broadly to refer to any segment of DNAassociated with a biological function. Thus, genes include codingsequences and/or the regulatory sequences required for their expression.Genes also include nonexpressed DNA segments that, for example, formrecognition sequences for other proteins. Genes can be obtained from avariety of sources, including cloning from a source of interest orsynthesizing from known or predicted sequence information, and mayinclude sequences designed to have desired parameters.

The term “isolated”, when applied to a nucleic acid or protein, denotesthat the nucleic acid or protein is essentially free of other cellularcomponents with which it is associated in the natural state. It ispreferably in a homogeneous state although it can be in either a dry oraqueous solution. Purity and homogeneity are typically determined usinganalytical chemistry techniques such as polyacrylamide gelelectrophoresis or high performance liquid chromatography. A proteinwhich is the predominant species present in a preparation issubstantially purified. In particular, an isolated gene is separatedfrom open reading frames which flank the gene and encode a protein otherthan the gene of interest. The term “purified” denotes that a nucleicacid or protein gives rise to essentially one band in an electrophoreticgel. Particularly, it means that the nucleic acid or protein is at leastabout 50% pure, more preferably at least about 85% pure, and mostpreferably at least about 99% pure.

The term “naturally-occurring” is used to describe an object that can befound in nature as distinct from being artificially produced by man. Forexample, a polypeptide or polynucleotide sequence that is present in anorganism (including viruses, bacteria, protozoa, insects, plants ormammalian tissue) that can be isolated from a source in nature and whichhas not been intentionally modified by man in the laboratory isnaturally-occurring.

The term “nucleic acid” refers to deoxyribonucleotides orribonucleotides and polymers thereof in either single- ordouble-stranded form. Unless specifically limited, the term encompassesnucleic acids containing known analogues of natural nucleotides whichhave similar binding properties as the reference nucleic acid and aremetabolized in a manner similar to naturally occurring nucleotides.Unless otherwise indicated, a particular nucleic acid sequence alsoimplicitly encompasses conservatively modified variants thereof (e.g.degenerate codon substitutions) and complementary sequences and as wellas the sequence explicitly indicated. Specifically, degenerate codonsubstitutions may be achieved by generating sequences in which the thirdposition of one or more selected (or all) codons is substituted withmixed-base and/or deoxyinosine residues (Batzer et al. (1991) NucleicAcid Res. 19: 5081; Ohtsuka et al. (1985) J. Biol. Chem. 260: 2605-2608;Cassol et al. (1992); Rossolini et al. (1994) Mol. Cell. Probes 8:91-98). The term nucleic acid is used interchangeably with gene, cDNA,and mRNA encoded by a gene.

“Nucleic acid derived from a gene” refers to a nucleic acid for whosesynthesis the gene, or a subsequence thereof, has ultimately served as atemplate. Thus, an mRNA, a cDNA reverse transcribed from an mRNA, an RNAtranscribed from that cDNA, a DNA amplified from the cDNA, an RNAtranscribed from the amplified DNA, etc., are all derived from the geneand detection of such derived products is indicative of the presenceand/or abundance of the original gene and/or gene transcript in asample.

A nucleic acid is “operably linked” when it is placed into a functionalrelationship with another nucleic acid sequence. For instance, apromoter or enhancer is operably linked to a coding sequence if itincreases the transcription of the coding sequence. Operably linkedmeans that the DNA sequences being linked are typically contiguous and,where necessary to join two protein coding regions, contiguous and inreading frame. However, since enhancers generally function whenseparated from the promoter by several kilobases and intronic sequencesmay be of variable lengths, some polynucleotide elements may be operablylinked but not contiguous.

A specific binding affinity between two molecules, for example, a ligandand a receptor, means a preferential binding of one molecule for anotherin a mixture of molecules. The binding of the molecules can beconsidered specific if the binding affinity is about 1×10⁴ M⁻¹ to about1×10⁶ M⁻¹ or greater.

The term “recombinant” when used with reference to a cell indicates thatthe cell replicates a heterologous nucleic acid, or expresses a peptideor protein encoded by a heterologous nucleic acid. Recombinant cells cancontain genes that are not found within the native (non-recombinant)form of the cell. Recombinant cells can also contain genes found in thenative form of the cell wherein the genes are modified and re-introducedinto the cell by artificial means. The term also encompasses cells thatcontain a nucleic acid endogenous to the cell that has been modifiedwithout removing the nucleic acid from the cell; such modificationsinclude those obtained by gene replacement, site-specific mutation, andrelated techniques.

A “recombinant expression cassette” or simply an “expression cassette”is a nucleic acid construct, generated recombinantly or synthetically,with nucleic acid elements that are capable of effecting expression of astructural gene in hosts compatible with such sequences. Expressioncassettes include at least promoters and optionally, transcriptiontermination signals. Typically, the recombinant expression cassetteincludes a nucleic acid to be transcribed (e.g., a nucleic acid encodinga desired polypeptide), and a promoter. Additional factors necessary orhelpful in effecting expression may also be used as described herein.For example, an expression cassette can also include nucleotidesequences that encode a signal sequence that directs secretion of anexpressed protein from the host cell. Transcription termination signals,enhancers, and other nucleic acid sequences that influence geneexpression, can also be included in an expression cassette.

A “recombinant polynucleotide” or a “recombinant polypeptide” is anon-naturally occurring polynucleotide or polypeptide that includesnucleic acid or amino acid sequences, respectively, from more than onesource nucleic acid or polypeptide, which source nucleic acid orpolypeptide can be a naturally occurring nucleic acid or polypeptide, orcan itself have been subjected to mutagenesis or other type ofmodification. The source polynucleotides or polypeptides from which thedifferent nucleic acid or amino acid sequences are derived are sometimeshomologous (i.e., have, or encode a polypeptide that encodes, the sameor a similar structure and/or function), and are often from differentisolates, serotypes, strains, species, of organism or from differentdisease states, for example.

The terms “identical” or percent “identity,” in the context of two ormore nucleic acid or polypeptide sequences, refer to two or moresequences or subsequences that are the same or have a specifiedpercentage of amino acid residues or nucleotides that are the same, whencompared and aligned for maximum correspondence, as measured using oneof the following sequence comparison algorithms or by visual inspection.

The phrase “substantially identical,” in the context of two nucleicacids or polypeptides, refers to two or more sequences or subsequencesthat have at least 60%, preferably 80%, most preferably 90-95%nucleotide or amino acid residue identity, when compared and aligned formaximum correspondence, as measured using one of the following sequencecomparison algorithms or by visual inspection. Preferably, thesubstantial identity exists over a region of the sequences that is atleast about 50 residues in length, more preferably over a region of atleast about 100 residues, and most preferably the sequences aresubstantially identical over at least about 150 residues. In someembodiments, the sequences are substantially identical over the entirelength of the coding regions.

For sequence comparison, typically one sequence acts as a referencesequence to which test sequences are compared. When using a sequencecomparison algorithm, test and reference sequences are input into acomputer, subsequence coordinates are designated, if necessary, andsequence algorithm program parameters are designated. The sequencecomparison algorithm then calculates the percent sequence identity forthe test sequence(s) relative to the reference sequence, based on thedesignated program parameters.

Optimal alignment of sequences for comparison can be conducted, e.g., bythe local homology algorithm of Smith & Waterman, Adv. Appl. Math. 2:482(1981), by the homology alignment algorithm of Needleman & Wunsch, J.Mol. Biol. 48:443 (1970), by the search for similarity method of Pearson& Lipman, Proc. Nat'l. Acad. Sci. USA 85:2444 (1988), by computerizedimplementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA inthe Wisconsin Genetics Software Package, Genetics Computer Group, 575Science Dr., Madison, Wis.), or by visual inspection (see generallyAusubel et al., infra).

One example of an algorithm that is suitable for determining percentsequence identity and sequence similarity is the BLAST algorithm, whichis described in Altschul et al., J. Mol. Biol. 215:403-410 (1990).Software for performing BLAST analyses is publicly available through theNational Center for Biotechnology Information(http://www.ncbi.nlm.nih.gov/). This algorithm involves firstidentifying high scoring sequence pairs (HSPs) by identifying shortwords of length W in the query sequence, which either match or satisfysome positive-valued threshold score T when aligned with a word of thesame length in a database sequence. T is referred to as the neighborhoodword score threshold (Altschul et al., supra). These initialneighborhood word hits act as seeds for initiating searches to findlonger HSPs containing them. The word hits are then extended in bothdirections along each sequence for as far as the cumulative alignmentscore can be increased. Cumulative scores are calculated using, fornucleotide sequences, the parameters M (reward score for a pair ofmatching residues; always >0) and N (penalty score for mismatchingresidues; always <0). For amino acid sequences, a scoring matrix is usedto calculate the cumulative score. Extension of the word hits in eachdirection are halted when: the cumulative alignment score falls off bythe quantity X from its maximum achieved value; the cumulative scoregoes to zero or below, due to the accumulation of one or morenegative-scoring residue alignments; or the end of either sequence isreached. The BLAST algorithm parameters W, T, and X determine thesensitivity and speed of the alignment. The BLASTN program (fornucleotide sequences) uses as defaults a wordlength (W) of 11, anexpectation (E) of 10, a cutoff of 100, M=5, N=−4, and a comparison ofboth strands. For amino acid sequences, the BLASTP program uses asdefaults a wordlength (W) of 3, an expectation (E) of 10, and theBLOSUM62 scoring matrix (see Henikoff & Henikoff (1989) Proc. Natl.Acad. Sci. USA 89:10915).

In addition to calculating percent sequence identity, the BLASTalgorithm also performs a statistical analysis of the similarity betweentwo sequences (see, e.g., Karlin & Altschul (1993) Proc. Nat'l. Acad.Sci. USA 90:5873-5787). One measure of similarity provided by the BLASTalgorithm is the smallest sum probability (P(N)), which provides anindication of the probability by which a match between two nucleotide oramino acid sequences would occur by chance. For example, a nucleic acidis considered similar to a reference sequence if the smallest sumprobability in a comparison of the test nucleic acid to the referencenucleic acid is less than about 0.1, more preferably less than about0.01, and most preferably less than about 0.001.

Another indication that two nucleic acid sequences are substantiallyidentical is that the two molecules hybridize to each other tinderstringent conditions. The phrase “hybridizing specifically to”, refersto the binding, duplexing, or hybridizing of a molecule only to aparticular nucleotide sequence under stringent conditions when thatsequence is present in a complex mixture (e.g., total cellular) DNA orRNA. “Bind(s) substantially” refers to complementary hybridizationbetween a probe nucleic acid and a target nucleic acid and embracesminor mismatches that can be accommodated by reducing the stringency ofthe hybridization media to achieve the desired detection of the targetpolynucleotide sequence.

“Stringent hybridization conditions” and “stringent hybridization washconditions” in the context of nucleic acid hybridization experimentssuch as Southern and northern hybridizations are sequence dependent, andare different under different environmental parameters. Longer sequenceshybridize specifically at higher temperatures. An extensive guide to thehybridization of nucleic acids is found in Tijssen (1993) LaboratoryTechniques in Biochemistry and Molecular Biology—Hybridization withNucleic Acid Probes part I chapter 2 “Overview of principles ofhybridization and the strategy of nucleic acid probe assays”, Elsevier,New York. Generally, highly stringent hybridization and wash conditionsare selected to be about 5° C. lower than the thermal melting point(T_(m)) for the specific sequence at a defined ionic strength and pH.Typically, under “stringent conditions” a probe will hybridize to itstarget subsequence, but to no other sequences.

The T_(m) is the temperature (under defined ionic strength and pH) atwhich 50% of the target sequence hybridizes to a perfectly matchedprobe. Very stringent conditions are selected to be equal to the T_(m)for a particular probe. An example of stringent hybridization conditionsfor hybridization of complementary nucleic acids which have more than100 complementary residues on a filter in a Southern or northern blot is50% formamide with 1 mg of heparin at 42° C., with the hybridizationbeing carried out overnight. An example of highly stringent washconditions is 0.15M NaCl at 72° C. for about 15 minutes. An example ofstringent wash conditions is a 0.2×SSC wash at 65° C. for 15 minutes(see, Sambrook, infra., for a description of SSC buffer). Often, a highstringency wash is preceded by a low stringency wash to removebackground probe signal. An example medium stringency wash for a duplexof, e.g., more than 100 nucleotides, is 1×SSC at 45° C. for 15 minutes.An example low stringency wash for a duplex of, e.g., more than 100nucleotides, is 4-6×SSC at 40° C. for 15 minutes. For short probes(e.g., about 10 to 50 nucleotides), stringent conditions typicallyinvolve salt concentrations of less than about 1.0 M Na⁺ ion, typicallyabout 0.01 to 1.0 M Na⁺ ion concentration (or other salts) at pH 7.0 to8.3, and the temperature is typically at least about 30° C. Stringentconditions can also be achieved with the addition of destabilizingagents such as formamide. In general, a signal to noise ratio of 2× (orhigher) than that observed for an unrelated probe in the particularhybridization assay indicates detection of a specific hybridization.Nucleic acids which do not hybridize to each other under stringentconditions are still substantially identical if the polypeptides whichthey encode are substantially identical. This occurs, e.g., when a copyof a nucleic acid is created using the maximum codon degeneracypermitted by the genetic code.

A further indication that two nucleic acid sequences or polypeptides aresubstantially identical is that the polypeptide encoded by the firstnucleic acid is immunologically cross reactive with, or specificallybinds to, the polypeptide encoded by the second nucleic acid. Thus, apolypeptide is typically substantially identical to a secondpolypeptide, for example, where the two peptides differ only byconservative substitutions.

The phrase “specifically (or selectively) binds to an antibody” or“specifically (or selectively) immunoreactive with”, when referring to aprotein or peptide, refers to a binding reaction which is determinativeof the presence of the protein, or an epitope from the protein, in thepresence of a heterogeneous population of proteins and other biologics.Thus, under designated immunoassay conditions, the specified antibodiesbind to a particular protein and do not bind in a significant amount toother proteins present in the sample. The antibodies raised against amultivalent antigenic polypeptide will generally bind to the proteinsfrom which one or more of the epitopes were obtained. Specific bindingto an antibody under such conditions may require an antibody that isselected for its specificity for a particular protein. A variety ofimmunoassay formats may be used to select antibodies specificallyimmunoreactive with a particular protein. For example, solid-phase ELISAimmunoassays, Western blots, or immunohistochemistry are routinely usedto select monoclonal antibodies specifically immunoreactive with aprotein. See Harlow and Lane (1988) Antibodies, A Laboratory Manual,Cold Spring Harbor Publications, New York “Harlow and Lane”), for adescription of immunoassay formats and conditions that can be used todetermine specific immunoreactivity. Typically a specific or selectivereaction will be at least twice background signal or noise and moretypically more than 10 to 100 times background.

“Conservatively modified variations” of a particular polynucleotidesequence refers to those polynucleotides that encode identical oressentially identical amino acid sequences, or where the polynucleotidedoes not encode an amino acid sequence, to essentially identicalsequences. Because of the degeneracy of the genetic code, a large numberof functionally identical nucleic acids encode any given polypeptide.For instance, the codons CGU, CGC, CGA, CGG, AGA, and AGG all encode theamino acid arginine. Thus, at every position where an arginine isspecified by a codon, the codon can be altered to any of thecorresponding codons described without altering the encoded polypeptide.Such nucleic acid variations are “silent variations,” which are onespecies of “conservatively modified variations.” Every polynucleotidesequence described herein which encodes a polypeptide also describesevery possible silent variation, except where otherwise noted. One ofskill will recognize that each codon in a nucleic acid (except AUG,which is ordinarily the only codon for methionine) can be modified toyield a functionally identical molecule by standard techniques.Accordingly, each “silent variation” of a nucleic acid which encodes apolypeptide is implicit in each described sequence.

Furthermore, one of skill will recognize that individual substitutions,deletions or additions which alter, add or delete a single amino acid ora small percentage of amino acids (typically less than 5%, moretypically less than 1%) in an encoded sequence are “conservativelymodified variations” where the alterations result in the substitution ofan amino acid with a chemically similar amino acid. Conservativesubstitution tables providing functionally similar amino acids are wellknown in the art. The following five groups each contain amino acidsthat are conservative substitutions for one another:

Aliphatic: Glycine (G), Alanine (A), Valine (V), Leucine (L), Isoleucine(I);

-   -   Aromatic: Phenylalanine (F), Tyrosine (Y), Tryptophan (W);    -   Sulfur-containing: Methionine (M), Cysteine (C);    -   Basic: Arginine (R), Lysine (K), Histidine (H);    -   Acidic: Aspartic acid (D), Glutamic acid (E), Asparagine (N),        Glutamine (Q). See also, Creighton (1984) Proteins, W.H. Freeman        and Company, for additional groupings of amino acids. In        addition, individual substitutions, deletions or additions which        alter, add or delete a single amino acid or a small percentage        of amino acids in an encoded sequence are also “conservatively        modified variations”.

A “subsequence” refers to a sequence of nucleic acids or amino acidsthat comprise a part of a longer sequence of nucleic acids or aminoacids (e.g., polypeptide) respectively.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

The present invention provides methods for obtaining polynucleotidesequences that, either directly or indirectly (i.e., through encoding apolypeptide), can modulate an immune response when present on a geneticvaccine vector. In another embodiment, the invention provides methodsfor optimizing the transport and presentation of antigens. The optimizedimmunomodulatory polynucleotides obtained using the methods of theinvention are particularly suited for use in conjunction with vaccines,including genetic vaccines. One of the advantages of genetic vaccines isthat one can incorporate genes encoding immunomodulatory molecules, suchas cytokines, costimulatory molecules, and molecules that improveantigen transport and presentation into the genetic vaccine vectors.This provides opportunities to modulate immune responses that areinduced against the antigens expressed by the genetic vaccines.

A. Creation of Recombinant Libraries

The invention involves creating recombinant libraries of polynucleotidesthat are then screened to identify those library members that exhibit adesired property. The recombinant libraries can be created using any ofvarious methods.

The substrate nucleic acids used for the recombination can varydepending upon the particular application. For example, where apolynucleotide that encodes a cytokine, chemokine, or other accessorymolecule is to be optimized, different forms of nucleic acids thatencode all or part of the cytokine, chemokine, or other accessorymolecule are subjected to recombination. The methods require at leasttwo variant forms of a starting substrate. The variant forms ofcandidate substrates can show substantial sequence or secondarystructural similarity with each other, but they should also differ in atleast two positions. The initial diversity between forms can be theresult of natural variation, e.g., the different variant forms(homologs) are obtained from different individuals or strains of anorganism (including geographic variants) or constitute related sequencesfrom the same organism (e.g., allelic variations). Alternatively, theinitial diversity can be induced, e.g., the second variant form can begenerated by error-prone transcription, such as an error-prone PCR oruse of a polymerase which lacks proof-reading activity (see Liao (1990)Gene 88:107-111), of the first variant form, or, by replication of thefirst form in a mutator strain (mutator host cells are discussed infurther detail below). The initial diversity between substrates isgreatly augmented in subsequent steps of recursive sequencerecombination.

Often, improvements are achieved after one round of recombination andselection. However, recursive sequence recombination can be employed toachieve still further improvements in a desired property. Sequencerecombination can be achieved in many different formats and permutationsof formats, as described in further detail below. These formats sharesome common principles. Recursive sequence recombination entailssuccessive cycles of recombination to generate molecular diversity. Thatis, one creates a family of nucleic acid molecules showing some sequenceidentity to each other but differing in the presence of mutations. Inany given cycle, recombination can occur in vivo or in vitro,intracellular or extracellular. Furthermore, diversity resulting fromrecombination can be augmented in any cycle by applying prior methods ofmutagenesis (e.g., error-prone PCR or cassette mutagenesis) to eitherthe substrates or products for recombination. In some instances, a newor improved property or characteristic can be achieved after only asingle cycle of in vivo or in vitro recombination, as when usingdifferent, variant forms of the sequence, as homologs from differentindividuals or strains of an organism, or related sequences from thesame organism, as allelic variations.

In a presently preferred embodiment, the recombinant libraries areprepared using DNA shuffling. The shuffling and screening or selectioncan be used to “evolve” individual genes, whole plasmids or viruses,multigene clusters, or even whole genomes (Stemmer (1995) Bio/Technology13:549-553). Reiterative cycles of recombination and screening/selectioncan be performed to further evolve the nucleic acids of interest. Suchtechniques do not require the extensive analysis and computationrequired by conventional methods for polypeptide engineering. Shufflingallows the recombination of large numbers of mutations in a minimumnumber of selection cycles, in contrast to traditional, pairwiserecombination events. Thus, the sequence recombination techniquesdescribed herein provide particular advantages in that they providerecombination between mutations in any or all of these, therebyproviding a very fast way of exploring the manner in which differentcombinations of mutations can affect a desired result. In someinstances, however, structural and/or functional information isavailable which, although not required for sequence recombination,provides opportunities for modification of the technique.

Exemplary formats and examples for sequence recombination, sometimesreferred to as DNA shuffling, evolution, or molecular breeding, havebeen described by the present inventors and co-workers in co-pendingapplications U.S. patent application Ser. No. 08/198,431, filed Feb. 17,1994, Serial No. PCT/US95/02126, filed, Feb. 17, 1995, Ser. No.08/425,684, filed Apr. 18, 1995, Ser. No. 08/537,874, filed October 30,1.995, Ser. No. 08/564,955, filed Nov. 30, 1995, Ser. No. 08/621,859,filed Mar. 25, 1996, Ser. No. 08/621,430, filed Mar. 25, 1996, SerialNo. PCT/US96/05480, filed Apr. 18, 1996, Ser. No. 08/650,400, filed May20, 1996, Ser. No. 08/675,502, filed Jul. 3, 1996, Ser. No. 08/721, 824,filed Sep. 27, 1996, Serial No. PCT/US97/17300, filed Sep. 26, 1997, andSerial No. PCT/US97/24239, filed Dec. 17, 1997; Stemmer, Science270:1510 (1995); Stemmer et al., Gene 164:49-53 (1995); Stemmer,Bio/Technology 13:549-553 (1995); Stemmer, Proc. Natl. Acad. Sci. U.S.A.91:10747-10751 (1994); Stemmer, Nature 370:389-391 (1994); Crameri etal., Nature Medicine 2(1):1-3 (1996); Crameri et al., NatureBiotechnology 14:315-319 (1996), each of which is incorporated byreference in its entirety for all purposes.

Other methods for obtaining recombinant polynucleotides and/or forobtaining diversity in nucleic acids used as the substrates forshuffling include, for example, homologous recombination(PCT/US98/05223; Publ. No. WO98/42727); oligonucleotide-directedmutagenesis (for review see, Smith, Ann. Rev. Genet. 19: 423-462 (1985);Botstein and Shortle, Science 229: 1193-1201 (1985); Carter, Biochem. J.237: 1-7 (1986); Kunkel, “The efficiency of oligonucleotide directedmutagenesis” in Nucleic acids & Molecular Biology, Eckstein and Lilley,eds., Springer Verlag, Berlin (1987)). Included among these methods areoligonucleotide-directed mutagenesis (Zoller and Smith, Nucl. Acids Res.10: 6487-6500 (1982), Methods in Enzymol. 100: 468-500 (1983), andMethods in Enzymol. 154: 329-350 (1987)) phosphothioate-modified DNAmutagenesis (Taylor et al., Nucl. Acids Res. 13: 8749-8764 (1985);Taylor et al., Nucl. Acids Res. 13: 8765-8787 (1985); Nakamaye andEckstein, Nucl. Acids Res. 14: 9679-9698 (1986); Sayers et al., Nucl.Acids Res. 16: 791-802 (1988); Sayers et al., Nucl. Acids Res. 16:803-814 (1988)), mutagenesis using uracil-containing templates (Kunkel,Proc. Nat'l. Acad. Sci. USA 82: 488-492 (1985) and Kunkel et al.,Methods in Enzymol. 154: 367-382)); mutagenesis using gapped duplex DNA(Kramer et al., Nucl. Acids Res. 12: 9441-9456 (1984); Kramer and Fritz,Methods in Enzymol. 154: 350-367 (1987); Kramer et al., Nucl. Acids Res.16: 7207 (1988)); and Fritz et al., Nucl. Acids Res. 16: 6987-6999(1988)). Additional suitable methods include point mismatch repair(Kramer et al., Cell 38: 879-887 (1984)), mutagenesis usingrepair-deficient host strains (Carter et al., Nucl. Acids Res. 13:4431-4443 (1985); Carter, Methods in Enzymol. 154: 382-403 (1987)),deletion mutagenesis (Eghtedarzadeh and Henikoff, Nucl. Acids Res. 14:5115 (1986)), restriction-selection and restriction-purification (Wellset al., Phil. Trans. R. Soc. Lond. A 317: 415-423 (1986)), mutagenesisby total gene synthesis (Nambiar et al., Science 223: 1299-1301 (1984);Sakamar and Khorana, Nucl. Acids Res. 14: 6361-6372 (1988); Wells etal., Gene 34: 315-323 (1985); and Grundstrom et al., Nucl. Acids Res.13: 3305-3316 (1985). Kits for mutagenesis are commercially available(e.g., Bio-Rad, Amersham International, Anglian Biotechnology).

B. Screening Methods

A recombination cycle is usually followed by at least one cycle ofscreening or selection for molecules having a desired property orcharacteristic. If a recombination cycle is performed in vitro, theproducts of recombination, i.e., recombinant segments, are sometimesintroduced into cells before the screening step. Recombinant segmentscan also be linked to an appropriate vector or other regulatorysequences before screening. Alternatively, products of recombinationgenerated in vitro are sometimes packaged as viruses before screening.If recombination is performed in vivo, recombination products cansometimes be screened in the cells in which recombination occurred. Inother applications, recombinant segments are extracted from the cells,and optionally packaged as viruses, before screening.

The nature of screening or selection depends on what property orcharacteristic is to be acquired or the property or characteristic forwhich improvement is sought, and many examples are discussed below. Itis not usually necessary to understand the molecular basis by whichparticular products of recombination (recombinant segments) haveacquired new or improved properties or characteristics relative to thestarting substrates. For example, a genetic vaccine vector can have manycomponent sequences each having a different intended role (e.g., codingsequence, regulatory sequences, targeting sequences,stability-conferring sequences, immunomodulatory sequences, sequencesaffecting antigen presentation, and sequences affecting integration).Each of these component sequences can be varied and recombinedsimultaneously. Screening/selection can then be performed, for example,for recombinant segments that have increased episomal maintenance in atarget cell without the need to attribute such improvement to any of theindividual component sequences of the vector.

Depending on the particular screening protocol used for a desiredproperty, initial round(s) of screening can sometimes be performed inbacterial cells due to high transfection efficiencies and ease ofculture. Later rounds, and other types of screening which are notamenable to screening in bacterial cells, are performed in mammaliancells to optimize recombinant segments for use in an environment closeto that of their intended use. Final rounds of screening can beperformed in the precise cell type of intended use (e.g., a humanantigen-presenting cell). In some instances, this cell can be obtainedfrom a patient to be treated with a view, for example, to minimizingproblems of immunogenicity in this patient.

The screening or selection step identifies a subpopulation ofrecombinant segments that have evolved toward acquisition of a new orimproved desired property or properties useful in genetic vaccination.Depending on the screen, the recombinant segments can be identified ascomponents of cells, components of viruses or in free form. More thanone round of screening or selection can be performed after each round ofrecombination.

If further improvement in a property is desired, at least one andusually a collection of recombinant segments surviving a first round ofscreening/selection are subject to a further round of recombination.These recombinant segments can be recombined with each other or withexogenous segments representing the original substrates or furthervariants thereof. Again, recombination can proceed in vitro or in vivo.If the previous screening step identifies desired recombinant segmentsas components of cells, the components can be subjected to furtherrecombination in vivo, or can be subjected to further recombination invitro, or can be isolated before performing a round of in vitrorecombination. Conversely, if the previous screening step identifiesdesired recombinant segments in naked form or as components of viruses,these segments can be introduced into cells to perform a round of invivo recombination. The second round of recombination, irrespective howperformed, generates further recombinant segments which encompassadditional diversity than is present in recombinant segments resultingfrom previous rounds.

The second round of recombination can be followed by a further round ofscreening/selection according to the principles discussed above for thefirst round. The stringency of screening/selection can be increasedbetween rounds. Also, the nature of the screen and the property beingscreened for can vary between rounds if improvement in more than oneproperty is desired or if acquiring more than one new property isdesired. Additional rounds of recombination and screening can then beperformed until the recombinant segments have sufficiently evolved toacquire the desired new or improved property or function.

Various screening methods for particular applications are describedherein. In several instances, screening involves expressing therecombinant peptides or polypeptides encoded by the recombinantpolynucleotides of the library as fusions with a protein that isdisplayed on the surface of a replicable genetic package. For example,phage display can be used. See, e.g, Cwirla et al., Proc. Natl. Acad.Sci. USA 87: 6378-6382 (1990); Devlin et al., Science 249: 404-406(1990), Scott & Smith, Science 249: 386-388 (1990); Ladner et al., U.S.Pat. No. 5,571,698. Other replicable genetic packages include, forexample, bacteria, eukaryotic viruses, yeast, and spores.

The genetic packages most frequently used for display libraries arebacteriophage, particularly filamentous phage, and especially phage M13,Fd and F1. Most work has involved inserting libraries encodingpolypeptides to be displayed into either gIII or gVIII of these phageforming a fusion protein. See, e.g., Dower, WO 91/19818; Devlin,WO91/18989; MacCafferty, WO 92/01047 (gene III); Huse, WO 92/06204;Kang, WO 92/18619 (gene VIII). Such a fusion protein comprises a signalsequence, usually but not necessarily, from the phage coat protein, apolypeptide to be displayed and either the gene III or gene VIII proteinor a fragment thereof. Exogenous coding sequences are often inserted ator near the N-terminus of gene III or gene VIII although other insertionsites are possible.

Eukaryotic viruses can be used to display polypeptides in an analogousmanner. For example, display of human heregulin fused to gp70 of Moloneymurine leukemia virus has been reported by Han et al., Proc. Natl. Acad.Sci. USA 92: 9747-9751 (1995). Spores can also be used as replicablegenetic packages. In this case, polypeptides are displayed from theouter surface of the spore. For example, spores from B. subtilis havebeen reported to be suitable. Sequences of coat proteins of these sporesare provided by Donovan et al., J. Mol. Biol. 196, 1-10 (1987). Cellscan also be used as replicable genetic packages. Polypeptides to bedisplayed are inserted into a gene encoding a cell protein that isexpressed on the cells surface. Bacterial cells including Salmonellatyphimurium, Bacillus subtilis, Pseudomonas aeruginosa, Vibrio cholerae,Klebsiella pneumonia, Neisseria gonorrhoeae, Neisseria meningitidis,Bacteroides nodosus, Moraxella bovis, and especially Escherichia coliare preferred. Details of outer surface proteins are discussed by Ladneret al., U.S. Pat. No. 5,571,698 and references cited therein. Forexample, the lamB protein of E. coli is suitable.

A basic concept of display methods that use phage or other replicablegenetic package is the establishment of a physical association betweenDNA encoding a polypeptide to be screened and the polypeptide. Thisphysical association is provided by the replicable genetic package,which displays a polypeptide as part of a capsid enclosing the genome ofthe phage or other package, wherein the polypeptide is encoded by thegenome. The establishment of a physical association between polypeptidesand their genetic material allows simultaneous mass screening of verylarge numbers of phage bearing different polypeptides. Phage displayinga polypeptide with affinity to a target, e.g., a receptor, bind to thetarget and these phage are enriched by affinity screening to the target.The identity of polypeptides displayed from these phage can bedetermined from their respective genomes. Using these methods apolypeptide identified as having a binding affinity for a desired targetcan then be synthesized in bulk by conventional means, or thepolynucleotide that encodes the peptide or polypeptide can be used aspart of a genetic vaccine.

C. Evolution of Improved Immunomodulatory Sequences

Cytokines can dramatically influence macrophage activation andT_(H)1/T_(H)2 cell differentiation, and thereby the outcome ofinfectious diseases. In addition, recent studies strongly suggest thatDNA itself can act as adjuvant by activating the cells of the immunesystem. Specifically, unmethylated CpG-rich DNA sequences were shown toenhance T_(H)1 cell differentiation, activate cytokine synthesis bymonocytes and induce proliferation of B lymphocytes. The invention thusprovides methods for enhancing the immunomodulatory properties ofgenetic vaccines (a) by evolving the stimulatory properties of DNAitself and (b) by evolving genes encoding cytokines and relatedmolecules that are involved in immune system regulation. These genes arethen used in genetic vaccine vectors.

Of particular interest are IFN-α and IL-12, which skew immune responsestowards a T helper 1 (T_(H)1) cell phenotype and, thereby, improve thehost's capacity to counteract pathogen invasions. Also provided aremethods of obtaining improved immunomodulatory nucleic acids that arecapable of inhibiting or enhancing activation, differentiation, oranergy of antigen-specific T cells. Because of the limited informationabout the structures and mechanisms that regulate these events,molecular breeding techniques of the invention provide much fastersolutions than rational design.

The methods of the invention typically involve the use of DNA shufflingor other methods to create a library of recombinant polynucleotides. Thelibrary is then screened to identify recombinant polynucleotides in thelibrary, when included in a genetic vaccine vector or administered inconjunction with a genetic vaccine, are capable of enhancing orotherwise altering an immune response induced by the vector. Thescreening step, in some embodiments, can involve introducing a geneticvaccine vector that includes the recombinant polynucleotides intomammalian cells and determining whether the cells, or culture mediumobtained by growing the cells, is capable of modulating an immuneresponse.

Optimized recombinant vector modules obtained through polynucleotiderecombination are useful not only as components of genetic vaccinevectors, but also for production of polypeptides, e.g., modifiedcytokines and the like, that can be administered to a mammal to enhanceor shift an immune response. Polynucleotide sequences obtained using theDNA shuffling methods of the invention can be used as a component of agenetic vaccine, or can be used for production of cytokines and otherimmunomodulatory polypeptides that are themselves used as therapeutic orprophylactic reagents. If desired, the sequence of the optimizedimmunomodulatory polypeptide-encoding polynucleotides can be determinedand the deduced amino acid sequence used to produce polypeptides usingmethods known to those of skill in the art.

1. Immunostimulatory DNA Sequences

The invention provides methods of obtaining polynucleotides that areimmunostimulatory when introduced into a mammal. Oligonucleotides thatcontain hexamers with a central CpG flanked by two 5′ purines (GpA orApA) and two 3′ pyrimidines (TpC or TpT) efficiently induce cytokinesynthesis and B cell proliferation (Krieg et al. (1995) Nature 374: 546;Klinman et al. (1996) Proc. Nat'l. Acad. Sci. USA 93: 2879; Pisetsky(1996) Immunity 5: 303-10) in vitro and act as adjuvants in vivo.Genetic vaccine vectors in which immunostimulatory sequence—(ISS)containing oligos are inserted have increased capacity to enhanceantigen-specific antibody responses after DNA vaccination. The minimallength of an ISS oligonucleotide for functional activity in vitro iseight (Klinman et al., supra.). Twenty-mers with three CG motifs werefound to be significantly more efficient in inducing cytokine synthesisthan a 15-mer with two CG motifs (Id.). GGGG tetrads have been suggestedto be involved in binding of DNA to cell surfaces (macrophages expressreceptors for example scavenger receptors, that bind DNA) (Pisetsky etal., supra.).

According to the invention, a library is generated by subjecting torecombination random DNA (e.g., fragments of human, murine, or othergenomic DNA), oligonucleotides that contain known ISS, poly A, C, G or Tsequences, or combinations thereof. The DNA, which includes at leastfirst and second forms which differ from each other in two or morenucleotides, are recombined to produce a library of recombinantpolynucleotides.

The library is then screened to identify those recombinantpolynucleotides that exhibit immunostimulatory properties. For example,the library can be screened for induction cytokine production in vitroupon introduction of the library into an appropriate cell type. Adiagram of this procedure is shown in FIG. 5. Among the cytokines thatcan be used as an indicator of immunostimulatory activity are, forexample, IL-2, IL-4, IL-5, IL-6, IL-10, IL-12, IL-13, IL-15, and IFN-γ.One can also test for changes in ratios of IL-4/IFN-γ, IL-4/IL-2,IL-5/IFN-γ, IL-5/IL-2, IL-13/IFN-γ, IL-13/IL-2. An alternative screeningmethod is the determination of the ability to induce proliferation ofcells involved in immune responses, such as B cells, T cells,monocytes/macrophages, total PBL, and the like. Other screens includedetecting induction of APC activation based on changes in expressionlevels of surface antigens, such as B7-1 (CD80), B7-2 (CD86), MHC classI and II, and CD14.

Other useful screens include identifying recombinant polynucleotidesthat induce T cell proliferation. Because ISS sequences induce B cellactivation, and because of several homologies between surface antigensexpressed by T cells and B cells, polynucleotides can be obtained thathave stimulatory activities on T cells.

Libraries of recombinant polynucleotides can also be screened forimproved CTL and antibody responses in vivo and for improved protectionfrom infection, cancer, allergy or autoimmunity. Recombinantpolynucleotides that exhibit the desired property can be recovered fromthe cell and, if further improvement is desired, the shuffling andscreening can be repeated. Optimized ISS sequences can used as anadjuvant separately from an actual vaccine, or the DNA sequence ofinterest can be fused to a genetic vaccine vector.

2. Cytokines, Chemokines, and Accessory Molecules

The invention also provides methods for obtaining optimized cytokines,cytokine antagonists, chemokines, and other accessory molecules thatdirect, inhibit, or enhance immune responses. For example, the methodsof the invention can be used to obtain genetic vaccines and otherreagents (e.g., optimized cytokines, and the like) that, whenadministered to a mammal, improve or alter an immune response. Theseoptimized immunomodulators are useful for treating infectious diseases,as well as other conditions such as inflammatory disorders, in anantigen non-specific manner.

For example, the methods of the invention can be used to developoptimized immunomodulatory molecules for treating allergies. Theoptimized immunomodulatory molecules can be used alone or in conjunctionwith antigen-specific genetic vaccines to prevent or treat allergy. Fourbasic mechanisms are available by which one can achieve specificimmunotherapy of allergy. First, one can administer a reagent thatcauses a decrease in allergen-specific T_(H)2 cells. Second, a reagentcan be administered that causes an increase in allergen-specific T_(H)1cells. Third, one can direct an increase in suppressive CD8⁺ T cells.Finally, allergy can be treated by inducing anergy of allergen-specificT cells. In this Example, cytokines are optimized using the methods ofthe invention to obtain reagents that are effective in achieving one ormore of these immunotherapeutic goals. The methods of the invention areused to obtain anti-allergic cytokines that have one or more propertiessuch as improved specific activity, improved secretion afterintroduction into target cells, are effective at a lower dose thannatural cytokines, and fewer side effects. Targets of particularinterest include interferon-α/γ, IL-10, IL-12, and antagonists of IL-4and IL-13.

The optimized immunomodulators, or optimized recombinant polynucleotidesthat encode the immunomodulators, can be administered alone, or incombination with other accessory molecules. Inclusion of optimalconcentrations of the appropriate molecules can enhance a desired immuneresponse, and/or direct the induction or repression of a particular typeof immune response. The polynucleotides that encode the optimizedmolecules can be included in a genetic vaccine vector, or the optimizedmolecules encoded by the genes can be administered as polypeptides.

In the methods of the invention, a library of recombinantpolynucleotides that encode immunomodulators is created by subjectingsubstrate nucleic acids to a recombination protocol, such as DNAshuffling or other method known to those of skill in the art. Thesubstrate nucleic acids are typically two or more forms of a nucleicacid that encodes an immunomodulator of interest.

Cytokines are among the immunomodulators that can be improved using themethods of the invention. Cytokine synthesis profiles play a crucialrole in the capacity of the host to counteract viral, bacterial andparasitic infections, and cytokines can dramatically influence theefficacy of genetic vaccines and the outcome of infectious diseases.Several cytokines, for example IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7,IL-8, IL-9, IL-10, IL-11, IL-12, IL-13, IL-14, IL-15, IL-16, IL-17,IL-18, G-CSF, GM-CSF, IFN-α, IFN-γ, TGF-β, TNF-α, TNF-β, IL-20 (MDA-7),and flt-3 ligand have been shown stimulate immune responses in vitro orin vivo. Immune functions that can be enhanced using appropriatecytokines include, for example, B cell proliferation, Ig synthesis, Igisotype switching, T cell proliferation and cytokine synthesis,differentiation of T_(H)1 and T_(H)2 cells, activation and proliferationof CTLs, activation and cytokine production bymonocytes/macrophages/dendritic cells, and differentiation of dendriticcells from monocytes/macrophages.

In some embodiments, the invention provides methods of obtainingoptimized immunomodulators that can direct an immune response towards aT_(H)1 or a T_(H)2 response. The ability to influence the direction ofimmune responses in this manner is of great importance in development ofgenetic vaccines. Altering the type of T_(H) response can fundamentallychange the outcome of an infectious disease. A high frequency of T_(H)1cells generally protects from lethal infections with intracellularpathogens, whereas a dominant T_(H)2 phenotype often results indisseminated, chronic infections. For example, in human, the T_(H)1phenotype is present in the tuberculoid (resistant) form of leprosy,while the T_(H)2 phenotype is found in lepromatous, multibacillary(susceptible) lesions (Yamamura et al. (1991) Science 254: 277).Late-stage AIDS patients have the T_(H)2 phenotype. Studies in familymembers indicate that survival from meningococcal septicemia depends onthe cytokine synthesis profile of PBL, with high IL-10 synthesis beingassociated with a high risk of lethal outcome and high TNF-α beingassociated with a low risk. Similar examples are found in mice. Forexample, BALB/c mice are susceptible to Leishmania major infection;these mice develop a disseminated fatal disease with a T_(H)2 phenotype.Treatment with anti-IL-4 monoclonal antibodies or with IL-12 induces aT_(H)1 response, resulting in healing. Anti-interferon-γ monoclonalantibodies exacerbate the disease. For some applications, it ispreferable to direct an immune response in the direction of a T_(H)2response. For example, where increased mucosal immunity is desired,including protective immunity, enhancing the T_(H)2 response can lead toincreased antibody production, particularly IgA.

T helper (T_(H)) cells are probably the most important regulators of theimmune system. T_(H) cells are divided into two subsets, based on theircytokine synthesis pattern (Mosmann and Coffman (1989) Adv. Immunol. 46:111). T_(H)1 cells produce high levels of the cytokines IL-2 and IFN-γand no or minimal levels of IL-4, IL-5 and IL-13. In contrast, T_(H)2cells produce high levels of IL-4, IL-5 and IL-13, and IL-2 and IFN-γproduction is minimal or absent. T_(H)1 cells activate macrophages,dendritic cells and augment the cytolytic activity of CD8⁺ cytotoxic Tlymphocytes and natural killer (NK) cells (Paul and Seder (1994) Cell76: 241), whereas T_(H)2 cells provide efficient help for B cells andalso mediate allergic responses due to the capacity of T_(H)2 cells toinduce IgE isotype switching and differentiation of B cells into IgEsecreting cells (Punnonen et al. (1993) Proc. Nat'l. Acad. Sci. USA 90:3730).

The screening methods for improved cytokines, chemokines, and otheraccessory molecules are generally based on identification of modifiedmolecules that exhibit improved specific activity on target cells thatare sensitive to the respective cytokine, chemokine, or other accessorymolecules. A library of recombinant cytokine, chemokine, or accessorymolecule nucleic acids can be expressed on phage or as purified proteinand tested using in vitro cell culture assays, for example. Importantly,when analyzing the recombinant nucleic acids as components of DNAvaccines, one can identify the most optimal DNA sequences (in additionto the functions of the protein products) in terms of theirimmunostimulatory properties, transfection efficiency, and theircapacity to improve the stabilities of the vectors. The identifiedoptimized recombinant nucleic acids can then be subjected to new roundsof shuffling and selection.

In one embodiment of the invention, cytokines are evolved that directdifferentiation of T_(H)1 cells. Because of their capacities to skewimmune responses towards a T_(H)1 phenotype, the genes encodinginterferon-α (IFN-α) and interleukin-12 (IL-12) are preferred substratesfor recombination and selection in order to obtain maximal specificactivity and capacity to act as adjuvants in genetic vaccinations. IFN-αis a particularly preferred target for optimization using the methods ofthe invention because of its effects on the immune system, tumor cellsgrowth and viral replication. Due to these activities, IFN-α was thefirst cytokine to be used in clinical practice. Today, IFN-α is used fora wide variety of applications, including several types of cancers andviral diseases. IFN-α also efficiently directs differentiation of humanT cells into T_(H)1 phenotype (Parronchi et al. (1992) J. Immunol. 149:2977). However, it has not been thoroughly investigated in vaccinationmodels, because, in contrast to human systems, it does not affect T_(H)1differentiation in mice. The species difference was recently explainedby data indicating that, like IL-12, IFN-α induces STAT4 activation inhuman cells but not in murine cells, and STAT4 has been shown to berequired in IL-12 mediated T_(H)1 differentiation (Thierfelder et al.(1996) Nature 382: 171).

Family DNA shuffling is a preferred method for optimizing IFN-α, usingas substrates the mammalian IFN-α genes, which are 85%-97% homologous.Greater than 10²⁶ distinct recombinants can be generated from thenatural diversity in these genes. To allow rapid parallel analysis ofrecombinant interferons, one can employ high throughput methods fortheir expression and biological assay as fusion proteins onbacteriophage. Recombinants with improved potency and selectivityprofiles are being selectively bred for improved activity. Variantswhich demonstrate improved binding to IFN-α receptors can be selectedfor further analysis using a screen for mutants with optimal capacity todirect T_(H)1 differentiation. More specifically, the capacities ofIFN-α mutants to induce IL-2 and IFN-γ production in in vitro human Tlymphocyte cultures can be studied by cytokine-specific ELISA andcytoplasmic cytokine staining and flow cytometry.

IL-12 is perhaps the most potent cytokine that directs T_(H)1 responses,and it has also been shown to act as an adjuvant and enhance T_(H)1responses following genetic vaccinations (Kim et al. (1997) J. Immunol.158: 816). IL-12 is both structurally and functionally a uniquecytokine. It is the only heterodimeric cytokine known to date, composedof a 35 kD light chain (p35) and a 40 kD heavy chain (p40) (Kobayashi etal (1989) J. Exp. Med. 170: 827; Stern et al. (1990) Proc. Nat'l. Acad.Sci. USA 87: 6808). Recently Lieschke et al. ((1997) Nature Biotech. 15:35) demonstrated that a fusion between p35 and p40 genes results in asingle gene that has activity comparable to that of the two genesexpressed separately. These data indicate that it is possible to shuffleIL-12 genes as one entity, which is beneficial in designing theshuffling protocol. Because of its T cell growth promoting activities,one can use normal human peripheral blood T cells in the selection ofthe most active IL-12 genes, enabling direct selection of IL-12 mutantswith the most potent activities on human T cells. IL-12 mutants can beexpressed in CHO cells, for example, and the ability of the supernatantsto induce T cell proliferation determined (FIG. 6). The concentrationsof IL-12 in the supernatants can be normalized based on a specific ELISAthat detects a tag fused to the shuffled IL-12 molecules.

Incorporation of evolved IFN-α and/or IL-12 genes into genetic vaccinevectors is expected to be safe. The safety of IFN-α has beendemonstrated in numerous clinical studies and in everyday hospitalpractice. A Phase II trial of IL-12 in the treatment of patients withrenal cell cancer resulted in several unexpected adverse effects (Taharaet al. (1995) Human Gene Therapy 6: 1607). However, IL-12 gene as acomponent of genetic vaccines aims at high local expression levels,whereas the levels observed in circulation are minimal compared to thoseobserved after systemic bolus injections. In addition, some of theadverse effects of systemic IL-12 treatments are likely to be related toits unusually long half-life (up to 48 hours in monkeys). DNA shufflingmay allow selection for a shorter half-life, thereby reducing thetoxicity even after high bolus doses.

In other cases, genetic vaccines that can induce T_(H)2 responses arepreferred, especially when improved antibody production is desired. Asan example, IL-4 has been shown to direct differentiation of T_(H)2cells (which produce high levels of IL-4, IL-5 and IL-13, and mediateallergic immune responses). Immune responses that are skewed towardsT_(H)2 phenotype are preferred when genetic vaccines are used toimmunize against autoimmune diseases prophylactically. T_(H)1 responsesare also preferred when the vaccines are used to treat and modulateexisting autoimmune responses, because autoreactive T cells aregenerally of T_(H)1 phenotype (Liblau et al. (1995) Immunol. Today16:34-38). IL-4 is also the most potent cytokine in induction of IgEsynthesis; IL-4 deficient mice are unable to produce IgE. Asthma andallergies are associated with an increased frequency of IL-4 producingcells, and are genetically linked to the locus encoding IL-4, which ison chromosome 5 (in close proximity to genes encoding IL-3, IL-5, IL-9,IL-13 and GM-CSF). IL-4, which is produced by activated T cells,basophils and mast cells, is a protein that has 153 amino acids and twopotential N-glycosylation sites. Human IL-4 is only approximately 50%identical to mouse IL-4, and IL-4 activity is species-specific. Inhuman, IL-13 has activities similar to those of IL-4, but IL-13 is lesspotent than IL-4 in inducing IgE synthesis. IL-4 is the only cytokineknown to direct T_(H)2 differentiation.

Improved IL-2 agonists are also useful in directing T_(H)2 celldifferentiation, whereas improved IL-4 antagonists can direct T_(H)1cell differentiation. Improved IL-4 agonists and antagonists can begenerated by shuffling of IL-4 or soluble IL-4 receptor. The IL-4receptor consists of an IL-4R α-chain (140 kD high-affinity bindingunit) and an IL-2R y-chain (these cytokine receptors share a commonγ-chain). The IL-4R α-chain is shared by IL-4 and IL-13 receptorcomplexes. Both IL-4 and IL-13 induce phosphorylation of the IL-4Rα-chain, but expression of IL-4R α-chain alone on transfectants is notsufficient to provide a functional IL-4R. Soluble IL-4 receptorcurrently in clinical trials for the treatment of allergies. Using theDNA shuffling methods of the invention, one can evolve a soluble IL-4receptor that has improved affinity for IL-4. Such receptors are usefulfor the treatment of asthma and other T_(H)2 cell mediated diseases,such as severe allergies. The shuffling reactions can take advantage ofnatural diversity present in cDNA libraries from activated T cells fromhuman and other primates. In a typical embodiment, a shuffled IL-4Rα-chain library is expressed on a phage, and mutants that bind to IL-4with improved affinity are identified. The biological activity of theselected mutants is then assayed using cell-based assays.

IL-2 and IL-15 are also of particular interest for use in geneticvaccines. IL-2 acts as a growth factor for activated B and T cells, andit also modulates the functions of NK-cells. IL-2 is predominantlyproduced by T_(H)1-like T cell clones, and, therefore, it is consideredmainly to function in delayed type hypersensitivity reactions. However,IL-2 also has potent, direct effects on proliferation and Ig-synthesisby B cells. The complex immunoregulatory properties of IL-2 arereflected in the phenotype of IL-2 deficient mice, which have highmortality at young age and multiple defects in their immune functionsincluding spontaneous development of inflammatory bowel disease. IL-15is a more recently identified cytokine produced by multiple cell types.IL-15 shares several, but not all, activities with IL-2. Both IL-2 andIL-15 induce B cell growth and differentiation. However, assuming thatIL-15 production in IL-2 deficient mice is normal, it is clear thatIL-15 cannot substitute for the function of IL-2 in vivo, since thesemice have multiple immunodeficiencies. IL-2 has been shown tosynergistically enhance IL-10-induced human Ig production in thepresence of anti-CD40 mAbs, but it antagonized the effects of IL-4. IL-2also enhances IL-4-dependent IgE synthesis by purified B cells. On theother hand, IL-2 was shown to inhibit IL-4-dependent murine IgG1 and IgEsynthesis both in vitro and in vivo. Similarly, IL-2 inhibitedIL-4-dependent human IgE synthesis by unfractionated human PBMC, but theeffects were less significant than those of IFN-α or IFN-γ. Due to theircapacities to activate both B and T cells, IL-2 and IL-15 are useful invaccinations. In fact, IL-2, as protein and as a component of geneticvaccines, has been shown to improve the efficacy of the vaccinations.Improving the specific activity and/or expression levels/kinetics ofIL-2 and IL-15 through use of the DNA shuffling methods of the inventionincreases the advantageous effects compared to wild-type IL-2 and IL-15.

Another cytokine of particular interest for optimization and use ingenetic vaccines according to the methods of the invention isinterleukin-6. IL-6 is a monocyte-derived cytokine that was originallydescribed as a B cell differentiation factor or B cell stimulatoryfactor-2 because of its ability to enhance Ig levels secreted byactivated B cells. IL-6 has also been shown to enhance IL-4-induced IgEsynthesis. It has also been suggested that IL-6 is an obligatory factorfor human IgE synthesis, because neutralizing-anti-IL-6 mAbs completelyblocked IL-4-induced IgE synthesis. IL-6 deficient mice have impairedcapacity to produce IgA. Because of its potent activities on thedifferentiation of B cells, IL-6 can enhance the levels of specificantibodies produced following vaccination. It is particularly useful asa component of DNA vaccines because high local concentrations can beachieved, thereby providing the most potent effects on the cellsadjacent to the transfected cells expressing the immunogenic antigen.IL-6 with improved specific activity and/or with improved expressionlevels, obtained by DNA shuffling, will have more beneficial effectsthan the wild-type IL-6.

Interleukin-8 is another example of a cytokine that, when modifiedaccording to the methods of the invention, is useful in geneticvaccines. IL-8 was originally identified as a monocyte-derivedneutrophil chemotactic and activating factor. Subsequently, IL-8 wasalso shown to be chemotactic for T cells and to activate basophilsresulting in enhanced histamine and leukotriene release from thesecells. Furthermore, IL-8 inhibits adhesion of neutrophils tocytokine-activated endothelial cell monolayers, and it protects thesecells from neutrophil-mediated damage. Therefore, endothelial cellderived IL-8 was suggested to attenuate inflammatory events occurring inthe proximity of blood vessel walls. IL-8 also modulates immunoglobulinproduction, and inhibits IL-4-induced IgG4 and IgE synthesis by bothunfractionated human PBMC and purified B cells in vitro. This inhibitoryeffect was independent of IFN-α, IFN-γ or prostaglandin E2. In addition,IL-8 inhibited spontaneous IgE synthesis by PBMC derived from atopicpatients. Due to its capacity to attract inflammatory cells, IL-8, likeother chemotactic agents, is useful in potentiating the functionalproperties of vaccines, including DNA vaccines (acting as an adjuvant).The beneficial effects of IL-8 can be improved by using the DNAshuffling methods of the invention to obtain IL-8 with improved specificactivity and/or with improved expression in target cells.

Interleukin-5, and antagonists thereof, can also be optimized using themethods of the invention for use in genetic vaccines. IL-5 is primarilyproduced by T_(H)2-type T cells and appears to play an important role inthe pathogenesis of allergic disorders because of its ability to induceeosinophilia. IL-5 acts as an eosinophil differentiation and survivalfactor in both mouse and man. Blocking IL-5 activity by use ofneutralizing monoclonal antibodies strongly inhibits pulmonaryeosinophilia and hyperactivity in mouse models, and IL-5 deficient micedo not develop eosinophilia. These data also suggest that IL-5antagonists may have therapeutic potential in the treatment of allergiceosinophilia.

IL-5 has also been shown to enhance both proliferation of, and Igsynthesis by, activated mouse and human B cells. However, other studiessuggested that IL-5 has no effect on proliferation of human B cells,whereas it activated eosinophils. IL-5 apparently is not crucial formaturation or differentiation of conventional B cells, because antibodyresponses in IL-5 deficient mice are normal. However, these mice have adevelopmental defect in their CD5⁺ B cells indicating that IL-5 isrequired for normal differentiation of this B cell subset in mice. Atsuboptimal concentrations of IL-4, IL-5 was shown to enhance IgEsynthesis by human B cells in vitro. Furthermore, a recent studysuggested that the effects of IL-5 on human B cells depend on the modeof B cell stimulation. IL-5 significantly enhanced IgM synthesis by Bcells stimulated with Moraxella catarrhalis. In addition IL-5 synergizedwith suboptimal concentrations of IL-2, but had no effect on Igsynthesis by SAC-activated B cells. Activated human B cells alsoexpressed IL-5 mRNA suggesting that IL-5 may also regulate B cellfunction, including IgE synthesis, by autocrine mechanisms.

The invention provides methods of evolving an IL-S antagonist thatefficiently binds to and neutralizes IL-5 or its receptor. Theseantagonists are useful as a component of vaccines used for prophylaxisand treatment of allergies. Nucleic acids encoding IL-5, for example,from human and other mammalian species, are shuffled and screened forbinding to immobilized IL-5R for the initial screening. Polypeptidesthat exhibit the desired effect in the initial screening assays can thenbe screened for the highest biological activity using assays such asinhibition of growth of IL-S dependent cells lines cultured in thepresence of recombinant wild-type IL-5. Alternatively, shuffled IL-5Rα-chains are screened for improved binding to IL-5.

Tumor necrosis factors (α and β) and their receptors are also suitabletargets for modification and use in genetic vaccines. TNF-α, which wasoriginally described as cachectin because of its ability to causenecrosis of tumors, is a 17 kDa protein that is produced in lowquantities by almost all cells in the human body following activation.TNF-α acts as an endogenous pyrogen and induces the synthesis of severalproinflammatory cytokines, stimulates the production of acute phaseproteins, and induces proliferation of fibroblasts. TNF-α plays a majorrole in the pathogenesis of endotoxin shock. A membrane-bound form ofTNF-α (mTNF-α), which is involved in interactions between B- andT-cells, is rapidly upregulated within four hours of T cell activation.mTNF-α plays a role in the polyclonal B cell activation observed inpatients infected with HIV. Monoclonal antibodies specific for mTNF-α orthe p55 TNF-α receptor strongly inhibit IgE synthesis induced byactivated CD4⁺ T cell clones or their membranes. Mice deficient for p55TNF-αR are resistant to endotoxic shock, and soluble TNF-AR preventsautoimmune diabetes mellitus in NOD mice. Phase III trials using sTNF-αRin the treatment of rheumatoid arthritis are in progress, afterpromising results obtained in the phase II trials.

The methods of the invention can be used to, for example, evolve asoluble TNF-αR that has improved affinity, and thus is capable of actingas an antagonist for TNF activity. Nucleic acids that encode TNF-αR andexhibit sequence diversity, such as the natural diversity observed incDNA libraries from activated T cells of human and other primates, areshuffled. The shuffled nucleic acids are expressed, e.g., on phage,after which mutants are selected that bind to TNF-α with improvedaffinity. If desired, the improved mutants can be subjected to furtherassays using biological activity, and the shuffled genes can besubjected to one or more rounds of shuffling and screening.

Another target of interest for application of the methods of theinvention is interferon-γ, and the evolution of antagonists of thiscytokine. The receptor for IFN-γ consists of a binding componentglycoprotein of 90 kD, a 228 amino acid extracellular portion, atransmembrane region, and a 222 amino acid intracellular region.Glycosylation is not required for functional activity. A single chainprovides high affinity binding (10⁻⁹-10¹⁰ M), but is not sufficient forsignaling. Receptor components dimerize upon ligand binding. The mouseIFN-γ receptor is 53% identical to that of mouse at the amino acidlevel. The human and mouse receptors only bind human and mouse IFN-γ,respectively. Vaccinia, cowpox and camelpox viruses have homologues ofsIFN-γR, which have relatively low amino acid sequence similarity(˜20%), but are capable of efficient neutralization of IFN-γ in vitro.These homologues bind human, bovine, rat (but not mouse) IFN-γ, and mayhave in vivo activity as IFN-γ antagonists. All eight cysteines areconserved in human, mouse, myxoma and Shope fibroma virus (6 in vacciniavirus) IFN-γR polypeptides, indicating similar 3-D structures. Anextracellular portion of mIFN-γR with a kD of 100-300 pM has beenexpressed in insect cells. Treatment of NZB/W mice (a mouse model ofhuman SLE) with msIFN-γ receptor (100 mg/three times a week i.p.)inhibits the onset of glomerulonephritis. All mice treated with sIFN-γor anti-IFN-γ mAbs were alive 4 weeks after the treatment wasdiscontinued, compared with 50% in a placebo group, and 78% ofIFN-γ-treated mice died.

The methods of the invention can be used to evolve soluble IFN-γRreceptor polypeptides with improved affinity, and to evolve IFN-γ withimproved specific activity and improved capacity to activate cellularimmune responses. In each case nucleic acids encoding the respectivepolypeptide, and which exhibit sequence diversity (e.g., that observedin cDNA libraries from activated T cells from human and other primates),are subjected to recombination and screened to identify thoserecombinant nucleic acids that encode a polypeptide having improvedactivity. In the case of shuffled IFN-γR, the library of shufflednucleic acids can be expressed on phage, which are screened to identifymutants that bind to IFN-γ with improved affinity. In the case of IFN-γ,the shuffled library is analyzed for improved specific activity andimproved activation of the immune system, for example, by usingactivation of monocytes/macrophages as an assay. The evolved IFN-γmolecules can improve the efficacy of vaccinations (e.g. when used asadjuvants).

Diseases that can be treated using high-affinity sIFN-γR polypeptidesobtained using the methods of the invention include, for example,multiple sclerosis, systemic lupus erythematosus (SLE), organ rejectionafter treatment, and graft versus host disease. Multiple sclerosis, forexample, is characterized by increased expression of IFN-γ in the brainof the patients, and increased production of IFN-γ by patients' T cellsin vitro. IFN-γ treatment has been shown to significantly exacerbate thedisease (in contrast to EAE in mice).

Transforming growth factor (TGF)-β is another cytokine that can beoptimized for use in genetic vaccines using the methods of theinvention. TGF-β has growth regulatory activities on essentially allcell types, and it has also been shown to have complex modulatoryeffects on the cells of the immune system. TGF-β inhibits proliferationof both B and T cells, and it also suppresses development of anddifferentiation of cytotoxic T cells and NK cells. TGF-β has been shownto direct IgA switching in both murine and human B cells. It was alsoshown to induce germline a transcription in murine and human B cells,supporting the conclusion that TGF-β can specifically induce IgAswitching.

Due to its capacity to direct IgA switching, TGF-β is useful as acomponent of DNA vaccines which aim at inducing potent mucosal immunity,e.g. vaccines for diarrhea. Also, because of its potentanti-proliferative effects TGF-β is useful as a component oftherapeutical cancer vaccines. TGF-β with improved specific activityand/or with improved expression levels/kinetics will have increasedbeneficial effects compared to the wild-type TGF-β.

Cytokines that can be optimized using the methods of the invention alsoinclude granulocyte colony stimulating factor (G-CSF) andgranulocyte/macrophage colony stimulating factor (GM-CSF). Thesecytokines induce differentiation of bone marrow stem cell intogranulocytes/macrophages. Administration of G-CSF and GM-CSFsignificantly improve recovery from bone marrow (BM) transplantation andradiotherapy, reducing infections and time the patients have to spend inhospitals. GM-CSF enhances antibody production following DNAvaccination. G-CSF is a 175 amino acid protein, while GM-CSF has 127amino acids. Human G-CSF is 73% identical at the amino acid level tomurine G-CSF and the two proteins show species cross-reactivity. G-CSFhas a homodimeric receptor (dimeric with kD of 200 pM, monomeric ˜2-4nM), and the receptor for GM-CSF is a three subunit complex. Cell linestransfected with cDNA encoding G-CSF R proliferate in response to G-CSF.Cell lines dependent of GM-CSF available (such as TF-1). G-CSF isnontoxic and is presently working very well as a drug. However, thetreatment is expensive, and more potent G-CSF might reduce the cost forpatients and to the health care. Treatments with these cytokines aretypically short-lasting and the patients are likely to never need thesame treatment again reducing likelihood of problems withimmunogenicity.

The methods of the invention are useful for evolving G-CSF and/or GM-CSFwhich have improved specific activity, as well as other polypeptidesthat have G-CSF and/or GM-CSF activity. G-CSF and/or GM-CSF nucleicacids having sequence diversity, e.g., those obtained from cDNAlibraries from diverse species, are shuffled to create a library ofshuffled G-CSF and/or GM-CSF genes. These libraries can be screened by,for example, picking colonies, transfecting the plasmids into a suitablehost cell (e.g., CHO cells), and assaying the supernatants usingreceptor-positive cell lines. Alternatively, phage display or relatedtechniques can be used, again using receptor-positive cell lines. Yetanother screening method involves transfecting the shuffled genes intoG-CSF/GM-CSF-dependent cell lines. The cells are grown one cell per welland/or at very low density in large flasks, and the cells that growfastest are selected. Shuffled genes from these cells are isolated; ifdesired, these genes can be used for additional rounds of shuffling andselection.

Ciliary neurotrophic factor (CNTF) is another suitable target forapplication of the methods of the invention. CNTF has 200 amino acidswhich exhibit 80% sequence identity between rat and rabbit CNTFpolypeptides. CNTF has IL-6-like inflammatory effects, and inducessynthesis of acute phase proteins. CNTF is a cytosolic protein whichbelongs to the IL-6/IL-11/LIF/oncostatin M-family, and becomesbiologically active only after becoming available either by cellularlesion or by an unknown release mechanism. CNTF is expressed bymyelinating Schwann cells, astrocytes and sciatic nerves. Structurally,CNTF is a dimeric protein, with a novel anti-parallel arrangement of thesubunits. Each subunit adopts a double crossover four-helix bundle fold,in which two helices contribute to the dimer interface. Lys-155 mutantslose activity, and some Glu-153 mutants have 5-10 higher biologicalactivity. The receptor for CNTF consists of a specific CNTF receptorchain, gp130, and a LIF-β receptor. The CNTFR α-chain lacks atransmembrane domain portion, instead being GPI-anchored. At highconcentration, CNTF can mediate CNTFR-independent responses. SolubleCNTFR binds CNTF and thereafter can bind to LIFR and induce signalingthrough gp130. CNTF enhances survival of several types of neurons, andprotects neurons in an animal model of Huntington disease (in contrastto NGF, neurotrophic factor, and neurotrophin-3). CNTF receptor knockoutmice-have severe motor neuron deficits at birth, and CNTF knockout miceexhibit such deficits postnatally. CNTF also reduces obesity in mousemodels. Decreased expression of CNTF is sometimes observed inpsychiatric patients. Phase I studies in patients with ALS (annualincidence ˜{fraction (1/100 000)}, 5% familiar cases, 90% die within 6years) found significant side effects after doses higher than 5mg/kg/day subcutaneously (including anorexia, weight loss, reactivationof herpes simplex virus (HSVI), cough, increased oral secretions).Antibodies against CNTF were detected in almost all patients, thusillustrating the need for alternative CNTF with different immunologicalproperties.

The recombination and screening methods of the invention can be used toobtain modified CNTF polypeptides that exhibit decreased immunogenicityin vivo; higher specific activity is also obtainable using the methods.Shuffling is conducted using nuclei acids encoding CNTF. In a preferredembodiment, an IL-6/LIF/(CNTF) hybrid is obtained by shuffling using anexcess of oligonucleotides that encode to the receptor binding sites ofCNTF. Phage display can then be used to test for lack of binding to theIL-6/LIF receptor. This initial screen is followed by a test for highaffinity binding to the CNTF receptor, and, if desired, functionalassays using CNTF responsive cell lines. The shuffled CNTF polypeptidescan be tested to identify those that exhibit reduced immunogenicity uponadministration to a mammal.

Another way in which the recombination and screening methods of theinvention can be used to optimize CNTF is to improve secretion of thepolypeptide. When a CNTF cDNA is operably linked to a leader sequence ofhNGF, only 35-40 percent of the total CNTF produced is secreted.

Target diseases for treatment with optimized CNTF, using either theshuffled gene in an expression vector as in DNA vaccines, or a purifiedprotein, include obesity, amyotrophic lateral sclerosis (ALS, LouGehrig's disease), diabetic neuropathy, stroke, and brain surgery.

Polynucleotides that encode chemokines can also be optimized using themethods of the invention and included in a genetic vaccine vector. Atleast three classes of chemokines are known, based on structure: Cchemokines (such as lymphotactin), C-C chemokines (such as MCP-1, MCP-2,MCP-3, MCP-4, MIP-1a, MIP-1b, RANTES), C-X-C chemokines (such as IL-8,SDF-1, ELR, Mig, IP10) (Premack and Schall (1996) Nature Med. 2: 1174).Chemokines can attract other cells that mediate immune and inflammatoryfunctions, thereby potentiating the immune response. Cells that areattracted by different types of chemokines include, for example,lymphocytes, monocytes and neutrophils. Generally, C-X-C chemokines arechemoattractants for neutrophils but not for monocytes, C-C chemokinesattract monocytes and lymphocytes but not neutrophils, C chemokineattracts lymphocytes.

Genetic vaccine vectors can also include optimized recombinantpolynucleotides that encode surface-bound accessory molecules, such asthose that are involved in modulation and potentiation of immuneresponses. These molecules, which include, for example, B7-1 (CD80),B7-2 (CD86), CD40, ligand for CD40, CTLA-4, CD28, and CD150 (SLAM), canbe subjected to DNA shuffling to obtain variants have altered and/orimproved activities.

Optimized recombinant polynucleotides that encode CD1 molecules are alsouseful in a genetic vaccine vector for certain applications. CD1 arenonpolymorphic molecules that are structurally and functionally relatedto MHC molecules. Importantly, CD1 has MHC-like activities, and it canfunction as an antigen presenting molecule (Porcelli (1995) Adv.Immunol. 59: 1). CD1 is highly expressed on dendritic cells, which arevery efficient antigen presenting cells. Simultaneous transfection oftarget cells with DNA vaccine vectors encoding CD1 and an antigen ofinterest is likely to boost the immune response. Because CD1 cells, incontrast to MHC molecules, exhibit limited allelic diversity in anoutbred population (Porcelli, supra.), large populations of individualswith different genetic backgrounds can be vaccinated with one CD1allele. The functional properties of CD1 molecules can be improved bythe DNA shuffling methods of the invention.

Optimized recombinant TAP genes and/or gene products can also beincluded in a genetic vaccine vector. TAP genes and their optimizationfor various purposes are discussed in more detail below. Moreover, heatshock proteins (HSP), such as HSP70, can also be evolved for improvedpresentation and processing of antigens. HSP70 has been shown to act asadjuvant for induction of CD8⁺ T cell activation and it enhancesimmunogenicity of specific antigenic peptides (Blachere et al. (1997) J.Exp. Med. 186:1315-22). When HSP70 is encoded by a genetic vaccinevector, it is likely to enhance presentation and processing of antigenicpeptides and thereby improve the efficacy of the genetic vaccines. DNAshuffling can be used to further improve the properties, includingadjuvant activity, of heat shock proteins, such as HSP70.

Recombinantly produced cytokine, chemokine, and accessory moleculepolypeptides, as well as antagonists of these molecules, can be used toinfluence the type of immune response to a given stimulus. However, theadministration of polypeptides sometimes has shortcomings, includingshort half life, high expense, difficult to store (must be stored at 4°C.), and a requirement for large volumes. Also, bolus injections cansometimes cause side effects. Administration of polynucleotides thatencode the recombinant cytokines or other molecules overcomes most orall of these problems. DNA, for example, can be prepared in high purity,is stable, temperature resistant, noninfectious, easy to manufacture. Inaddition, polynucleotide-mediated administration of cytokines canprovide long-lasting, consistent expression, and administration ofpolynucleotides in general is regarded as being safe.

The functions of cytokines, chemokines and accessory molecules areredundant and pleiotropic, and therefore can be difficult to determinewhich cytokines or cytokine combinations are the most potent in inducingand enhancing antigen specific immune responses following vaccination.Furthermore, the most useful combination of cytokines and accessorymolecules is typically different depending on the type of immuneresponse that is desired following vaccination. As an example, IL-4 hasbeen shown to direct differentiation of T_(H)2 cells (which produce highlevels of IL-4, IL-5 and IL-13, and mediate allergic immune responses),whereas IFN-γ and IL-12 direct differentiation of T_(H)1 cells (whichproduce high levels of IL-2 and IFN-γ), and mediate delayed type immuneresponses. Moreover, the most useful combination of cytokines andaccessory molecules is also likely to depend on the antigen used in thevaccination. The invention provides a solution to this problem ofobtaining an optimized genetic vaccine cocktail. Different combinationsof cytokines, chemokines and accessory molecules are assembled intovectors using the methods described herein. These vectors are thenscreened for their capacity to induce immune responses in vivo and invitro.

Large libraries of vectors, generated by gene shuffling andcombinatorial molecular-biology, are screened for maximal capacity todirect immune responses towards, for example, a T_(H)1 or T_(H)2phenotype, as desired. A library of different vectors can be generatedby assembling different evolved promoters, (evolved) cytokines,(evolved) cytokine antagonists, (evolved) chemokines, (evolved)accessory molecules and immunostimulatory sequences, each of which canbe prepared using methods described herein. DNA sequences and compoundsthat facilitate the transfection and expression can be included. If thepathogen(s) is known, specific DNA sequences encoding immunogenicantigens from the pathogen can be incorporated into these vectorsproviding protective immunity against the pathogen(s) (as in geneticvaccines).

Initial screening is preferably carried out in vitro. For example, thelibrary can be introduced into cells which are tested for ability toinduce differentiation of T cells capable of producing cytokines thatare indicative of the type of immune response desired.

For a T_(H)1 response, for example, the library is screened to identifyrecombinant polynucleotides that are capable of inducing T cells toproduce IL-2 and IFN-γ, while screening for induction of T cellproduction of IL-4, IL-5, and IL-13 is performed to identify recombinantpolynucleotides that favor a T_(H)2 response.

Screening can also be conducted in vivo, using animal models. Forexample, vectors produced using the methods of the invention can betested for ability to protect against a lethal infection. Anotherscreening method involves injection of Leishmania major parasites intofootpads of BALB/c mice (nonhealer). Pools of plasmids are injectedi.v., i.p. or into footpads of these mice and the size of the footpadswelling is followed. Yet another in vivo screening method involvesdetection of IgE levels after infection with Nippostrongylusbrasiliensis. High levels indicate a T_(H)2 response, while low levelsof IgE indicate a T_(H)1 response.

Successful results in animal models are easy to verify in humans. Invitro screening can be conducted to test for human T_(H)1 or T_(H)2phenotype, or for other desired immune response. Vectors can also betested for ability to induce protection against infection in humans.

Because the principles of immune functions are similar in a wide varietyof infections, immunostimulating DNA vaccine vectors may not only beuseful in the treatment of a number of infectious diseases but also inprevention of the infections, when the vectors are delivered to thesites of the entry of the pathogen (e.g., the lung or gut).

3. Agonists or Antagonists of Cellular Receptors

The invention also provides methods for obtaining optimized recombinantpolynucleotides that encode a peptide or polypeptide that can interactwith a cellular receptor that is involved in mediating an immuneresponse. The optimized recombinant polynucleotides can act as anagonist or an antagonist of the receptor.

Cytokine antagonists can be used as components of genetic vaccinecocktails. Blocking immunosuppressive cytokines, rather than addingsingle proinflammatory cytokines, is likely to potentiate the immuneresponse in a more general manner, because several pathways arepotentiated at the same time. By appropriate choice of antagonist, onecan tailor the immune response induced by a genetic vaccine in order toobtain the response that is most effective in achieving the desiredeffect. Antagonists against any cytokine can be used as appropriate;particular cytokines of interest for blocking include, for example,IL-4, IL-13, IL-10, and the like.

The invention provides methods of obtaining cytokine antagonists thatexhibit greater effectiveness in blocking the action of the respectivecytokine. Polynucleotides that encode improved cytokine antagonists canbe obtained by using gene shuffling to generate a recombinant library ofpolynucleotides which are then screened to identify those that encode animproved antagonist. As substrates for the DNA shuffling, one can use,for example, polynucleotides that encode receptors for the respectivecytokine. At least two forms of the substrate will be present in therecombination reaction, with each form differing from the other in atleast one nucleotide position. In a preferred embodiment, the differentforms of the polynucleotide are homologous cytokine receptor genes fromdifferent organisms. The resulting library of recombinantpolynucleotides is then screened to identify those that encode cytokineantagonists with the desired affinity and biological activity.

As one example of the type of effect that one can achieve by including acytokine antagonist in a genetic vaccine cocktail, as well as how theeffect can be improved using the DNA shuffling methods of the invention,IL-10 is discussed. The same rationale can be applied to obtaining andusing antagonists of other cytokines. Interleukin-10 (IL-10) is perhapsthe most potent anti-inflammatory cytokine known to date. IL-10 inhibitsa number of pathways that potentiate inflammatory responses. Thebiological activities of IL-10 include inhibition of MHC class IIexpression on monocytes, inhibition of production of IL-1, IL-6, IL-12,TNF-α by monocytes/macrophages, and inhibition of proliferation and IL-2production by T lymphocytes. The significance of IL-10 as a regulatorymolecule of immune and inflammatory responses was clearly demonstratedin IL-10 deficient mice. These mice are growth-retarded, anemic andspontaneously develop an inflammatory bowel disease (Kuhn et al. (1993)Cell 75: 263). In addition, both innate and acquired immunity toListeria monocytogenes were shown to be elevated in IL-10 deficient mice(Dai et al. (1997) J. Immunol. 158: 2259). It has also been suggestedthat genetic differences in the levels of IL-10 production may affectthe risk of patients to die from complications meningococcal infection.Families with high IL-10 production had 20-fold increased risk of fataloutcome of meningococcal disease (Westendorp et al. (1997) Lancet 349:170).

IL-10 has been shown to activate normal and malignant B cells in vitro,but it does not appear to be a major growth promoting cytokine fornormal B cells in vivo, because IL-10 deficient mice have normal levelsof B lymphocytes and Ig in their circulation. In fact, there is evidencethat IL-10 can indirectly downregulate B cell function throughinhibition of the accessory cell function of monocytes. However, IL-10appears to play a role in the growth and expansion of malignant B cells.Anti-IL-10 monoclonal antibodies and IL-10 antisense oligonucleotideshave been shown to inhibit transformation of B cells by EBV in vitro. Inaddition, B cell lymphomas are associated with EBV and most EBV⁺lymphomas produce high levels of IL-10, which is derived both from thehuman gene and the homologue of IL-10 encoded by EBV. AIDS-related Bcell lymphomas also secrete high levels of IL-10. Furthermore, patientswith detectable serum IL-10 at the time of diagnosis ofintermediate/high-grade non-Hodgkin's lymphoma have short survival,further suggesting a role for IL-10 in the pathogenesis of B cellmalignancies.

Antagonizing IL-10 in vivo can be beneficial in several infectious andmalignant diseases, and in vaccination. The effect of blocking of IL-10is an enhancement of immune responses that is independent of thespecificity of the response. This is useful in vaccinations and in thetreatment of serious infectious diseases. Moreover, an IL-10 antagonistis useful in the treatment of B cell malignancies which exhibitoverproduction of IL-10 and viral IL-10, and it may also be useful inboosting general anti-tumor immune response in cancer patients.Combining an IL-10 antagonist with gene therapy vectors may be useful ingene therapy of tumor cells in order to obtain maximal immune responseagainst the tumor cells. If shuffling of IL-10 results in IL-10 withimproved specific activity, this IL-10 molecule would have potential inthe treatment of autoimmune diseases and inflammatory bowel diseases.IL-10 with improved specific activity may also be useful as a componentof gene therapy vectors in reducing the immune response against vectorswhich are recognized by memory cells and it may also reduce theimmunogenicity of these vectors.

An antagonist of IL-10 has been made by generating a soluble form ofIL-10 receptor (sIL-10R; Tan et al. (1995) J. Biol. Chem. 270: 12906).However, sIL-10R binds IL-10 with Kd of 560 pM, whereas the wild-type,surface-bound receptor has affinity of 35-200 pM. Consequently, 150-foldmolar excess of sIL-10R is required for half-maximal inhibition ofbiological function of IL-10. Moreover, affinity of viral IL-10 (IL-10homologue encoded by Epstein-Barr virus) to sIL-10R is more than 1000fold less than that of hIL-10, and in some situations, such as whentreating EBV-associated B cell malignancies, it may be beneficial if onecan also block the function of viral IL-10. Taken together, this solubleform of IL-10R is unlikely to be effective in antagonizing IL-10 invivo.

To obtain an IL-10 antagonist that has sufficient affinity andantagonistic activity to function in vivo, DNA shuffling can beperformed using polynucleotides that encode IL-10 receptor. IL-10receptor with higher than normal affinity will function as an IL-10antagonist, because it strongly reduces the amount of IL-10 availablefor binding to functional, wild-type IL-10R. In a preferred embodiment,IL-10R is shuffled using homologous cDNAs encoding IL-10R derived fromhuman and other mammalian species. An alignment of human and mouse IL-10receptor sequences is shown in FIG. 14 to illustrate the feasibility offamily DNA shuffling when evolving IL-10 receptors with improvedaffinity. A phage library of IL-10 receptor recombinants can be screenedfor improved binding of shuffled IL-10R to human or viral IL-10.Wild-type IL-10 and/or viral IL-10 are added at increasingconcentrations to demand for higher affinity. Phage bound to IL-10 canbe recovered using anti-IL-10 monoclonal antibodies. If desired, theshuffling can be repeated one or more times, after which the evolvedsoluble IL-10R is analyzed in functional assays for its capacity toneutralize the biological activities of IL-10/viral IL-10. Morespecifically, evolved soluble IL-10R is studied for its capacity toblock the inhibitory effects of IL-10 on cytokine synthesis and MHCclass II expression by monocytes, proliferation by T cells, and for itscapacity to inhibit the enhancing effects of IL-10 on proliferation of Bcells activated by anti-CD40 monoclonal antibodies.

An IL-10 antagonist can also be generated by evolving IL-10 to obtainvariants that bind to IL-10R with higher than wild-type affinity, butwithout receptor activation. The advantage of this approach is that onecan evolve an IL-10 molecule with improved specific activity using thesame methods. In a preferred embodiment, IL-10 is shuffled usinghomologous cDNAs encoding IL-10 derived from human and other mammalianspecies. In addition, a gene encoding viral IL-10 can be included in theshuffling. A library of IL-10 recombinants is screened for improvedbinding to human IL-10 receptor. Library members bound to IL-10R can berecovered by anti-IL-10R monoclonal antibodies. This screening protocolis likely to result in IL-10 molecules with both antagonistic andagonistic activities. Because initial screen demands for higheraffinity, a proportion of the agonists are likely to have improvedspecific activity when compared to wild-type human IL-10. The functionalproperties of the mutant IL-10 molecules are determined in biologicalassays similar to those described above for ultrahigh-affinity IL-10receptors (cytokine synthesis and MHC class II expression by monocytes,proliferation of B and T cells). An antagonistic IL-4 mutant has beenpreviously generated illustrating the general feasibility of theapproach (Kruse et al. (1992) EMBO J. 11: 3237-3244). One amino acidmutation in IL-4 resulted in a molecule that efficiently binds to IL-4Rα-chain but has minimal IL-4-like agonistic activity.

Another example of an IL-10 antagonist is IL-20/mda-7, which is a 206amino acid secreted protein. This protein was originally characterizedas mda-7, which is a melanoma cell-derived negative regulator of tumorcell growth (Jiang et al. (1995) Oncogene 11: 2477; (1996) Proc. Nat'l.Acad. Sci. USA 93: 9160). IL-20/mda-7 is structurally related to IL-10,and it antagonizes several functions of IL-10 (Abstract of the 13thEuropean Immunology Meeting, Amsterdam, 22-25 Jun. 1997). In contrast toIL-10, IL-20/mda-7 enhances expression of CD80 (B7-1) and CD86 (B7-2) onhuman monocytes and it upregulates production of TNF-α and IL-6.IL-20/mda-7 also enhances production of IFN-γ by PHA-activated PBMC. Theinvention provides methods of improving genetic vaccines byincorporation of IL-20/mda-7 genes into the genetic vaccine vectors. Themethods of the invention can be used to obtain IL-20/mda-7 variants thatexhibit improved ability to antagonize IL-10 activity.

When a cytokine antagonist is used as a component of DNA vaccine or genetherapy vectors, maximal local effect is desirable. Therefore, inaddition to a soluble form of a cytokine antagonist, a transmembraneform of the antagonist can be generated. The soluble form can be givenin purified polypeptide form to patients by, for example, intravenousinjection. Alternatively, a polynucleotide encoding the cytokineantagonist can be used as a component as a component of a geneticvaccine or a gene therapy vector. In this case, either or both of thesoluble and transmembrane forms can be used. Where both soluble andtransmembrane forms of the antagonist are encoded by the same vector,the target cells express both forms, resulting in maximal inhibition ofcytokine function on the target cell surface and in their immediatevicinity.

The peptides or polypeptides obtained using these methods can substitutefor the natural ligands of the receptors, such as cytokines or othercostimulatory molecules in their ability to exert an effect on theimmune system via the receptor. A potential disadvantage ofadministering cytokines or other costimulatory molecules themselves isthat an autoimmune reaction could be induced against the naturalmolecule, either due to breaking tolerance (if using a natural cytokineor other molecule) or by inducing cross-reactive immunity (humoral orcellular) when using related but distinct molecules. Through using themethods of the invention, one can obtain agonists or antagonists thatavoid these potential drawbacks. For example, one can use relativelysmall peptides as agonists that can mimic the activity of the naturalimmunomodulator, or antagonize the activity, without inducingcross-reactive immunity to the natural molecule. In a presentlypreferred embodiment, the optimized agonist or antagonist obtained usingthe methods of the invention is about 50 amino acids or length or less,more preferably about 30 amino acids or less, and most preferably isabout 20 amino acids in length, or less. The agonist or antagonistpeptide is preferably at least about 4 amino acids in length, and morepreferably at least about 8 amino acids in length. Polynucleotides thatflank the coding sequence of the mimetic peptide can also be optimizedusing the methods of the invention in order to optimize the expression,conformation, or activity of the mimetic peptide.

The optimized agonist or antagonist peptides or polypeptides areobtained by generating a library of recombinant polynucleotides andscreening the library to identify those that encode a peptide orpolypeptide that exhibits an enhanced ability to modulate an immuneresponse. The library can be produced using methods such as DNAshuffling or other methods described herein or otherwise known to thoseof skill in the art. Screening is conveniently conducted by expressingthe peptides encoded by the library members on the surface of apopulation of replicable genetic packages and identifying those membersthat bind to a target of interest, e.g., a receptor.

The optimized recombinant polynucleotides that are obtained using themethods of the invention can be used in several ways. For example, thepolynucleotide can be placed in a genetic vaccine vector, under thecontrol of appropriate expression control sequences, so that the mimeticpeptide is expressed upon introduction of the vector into a mammal. Ifdesired, the polynucleotide can be placed in the vector embedded in thecoding sequence of the surface protein (e.g., geneIII or geneVIII) inorder to preserve the conformation of the mimetic. Alternatively, themimetic-encoding polynucleotide can be inserted directly into theantigen-encoding sequence of the genetic vaccine to form a codingsequence for a “mimotope-on-antigen” structure. The polynucleotide thatencodes the mimotope-on-antigen structure can be used within a geneticvaccine, or can be used to express a protein that is itself administeredas a vaccine. As one example of this type of application, a codingsequence of a mimetic peptide is introduced into a polynucleotide thatencodes the “M-loop” of the hepatitis B surface antigen (HBsAg) protein.The M-loop is a six amino acid peptide sequence bounded by cysteineresidues, which is found at amino acids 139-147 (numbering within the Sprotein sequence). The M-loop in the natural HBsAg protein is recognizedby the monoclonal antibody RFHB7 (Chen et al., Proc. Nat'l. Acad. Sci.USA, 93: 1997-2001 (1996)). According to Chen et al., the M-loop formsan epitope of the HBsAg that is non-overlapping and separate from atleast four other HBsAg epitopes.

Because of the probable Cys-Cys disulfide bond in this hydrophilic partof the protein, amino acids 139-147 are likely in a cyclic conformation.This structure is therefore similar to that found in the regions of thefilamentous phage proteins pIII and pVIII where mimotope sequences areplaced. Therefore, one can insert a mimotope obtained using the methodsof the invention into this region of the HBsAg amino acid sequence.

The chemokine receptor CCR6 is an example of a suitable target for apeptide mimetic obtained using the methods. The CCR6 receptor is a7-transmembrane domain protein (Dieu et al., Biochem. Biophys. Res.Comm. 236: 212-217 (1997) and J. Biol. Chem. 272: 14893-14898 (1997))that is involved in the chemoattraction of immature dendritic cells,which are found in the blood and migrate to sites of antigen uptake(Dieu et al., J. Exp. Med. 188: 373-386 (1998)). CCR6 binds thechemokine MIP-3α, so a mimetic peptide that is capable of activatingCCR6 can provide a further chemoattractant function to a given antigenand thus promote uptake by dendritic cells after immunization with theantigen antigen-mimetic fusion or a DNA vector that expresses theantigen.

Another application of this method of the invention is to obtainmolecules that can act as an agonist for the macrophage scavengerreceptor (MSR; see, Wloch et al., Hum. Gene Ther. 9: 1439-1447 (1998)).The MSR is involved in mediating the effects of variousimmunomodulators. Among these are bacterial DNA, including the plasmidsused in DNA vaccination, and oligonucleotides, which are often potentimmunostimulators. Oligonucleotides of certain chemical structure (e.g.,phosphothio-oligonucleotides) are particularly potent, while bacterialor plasmid DNA must be used in relatively large quantities to produce aneffect. Also mediated by the MSR is the ability of oligonucleotides thatcontain dG residues to stimulate B cells and enhance the activity ofimmunostimulatory CpG motifs, and of lipopolysaccharides to activatemacrophages. Some of these activities are toxic. Each of theseimmunomodulators, along with a variety of polyanionic ligands, binds tothe MSR. The methods of the invention can be used to obtain mimetics ofone or more of these immunomodulators that bind to the MSR with highaffinity but are devoid of toxic properties. Such mimetic peptides areuseful as immunostimulators or adjuvants.

The MSR is a trimeric integral membrane glycoprotein. The threeextracellular C-terminal cysteine-rich regions are connected to thetransmembrane domain by a fibrous region that is composed of ana-helical coil and a collagen-like triple helix (see, Kodama et al.,Nature 343: 531-535 (1990)). Therefore, screening of the library ofrecombinant polynucleotides can be accomplished by expressing theextracellular receptor structure and artificially attaching it toplastic surfaces. The libraries can be expressed, e.g., by phagedisplay, and screened to identify those that bind to the receptors withhigh affinity. The optimized recombinant polynucleotides identified bythis method can be incorporated into antigen-encoding sequences toevaluate their modulatory effect on the immune response.

4. Costimulatory Molecules Capable of Inhibiting or EnhancingActivation, Differentiation, or Anergy of Antigen-Specific T Cells

Also provided are methods of obtaining optimized recombinantpolynucleotides that, when expressed, are capable of inhibiting orenhancing the activation, differentiation, or anergy of antigen-specificT cells. T cell activation is initiated when T cells recognize theirspecific antigenic peptides in the context of MHC molecules on theplasma membrane of antigen presenting cells (APC), such as monocytes,dendritic cells (DC), Langerhans cells or B cells. Activation of CD4⁺ Tcells requires recognition by the T cell receptor (TCR) of an antigenicpeptide in the context of MHC class II molecules, whereas CD8⁺ T cellsrecognize peptides in the context of MHC class I molecules. Importantly,however, recognition of the antigenic peptides is not sufficient forinduction of T cell proliferation and cytokine synthesis. An additionalcostimulatory signal, “the second signal”, is required. Thecostimulatory signal is mediated via CD28, which binds to its ligandsB7-1 (CD80) or B7-2 (CD86), typically expressed on the antigenpresenting cells. In the absence of the costimulatory signal, no T cellactivation occurs, or T cells are rendered anergic. In addition to CD28,CTLA-4 (CD152) also functions as a ligand for B7-1 and B7-2. However, incontrast to CD28, CTLA-4 mediates a negative regulatory signal to Tcells and/or to induce anergy and tolerance (Walunas et al. (1994)Immunity 1: 405; Karandikar et al. (1996) J. Exp. Med. 184: 783).

B7-1 and B7-2 have been shown to be able to regulate severalimmunological responses, and they have been implicated to be ofimportance in the immune regulation in vaccinations, allergy,autoimmunity and cancer. Gene therapy and genetic vaccine vectorsexpressing B7-1 and/or B7-2 have also been shown to have therapeuticpotential in the treatment of the above mentioned diseases and inimproving the efficacy of genetic vaccines.

FIG. 10 illustrates interaction of APC and CD4⁺ T cells, but the sameprinciple is true with CD8⁺ T cells, with the exception that the T cellsrecognize the antigenic peptides in the context of MHC class Imolecules. Both B7-1 and B7-2 bind to CD28 and CTLA-4, even though thesequence similarities between these four molecules are very limited(20-30%). It is desirable to obtain mutations in B7-1 and B7-2 that onlyinfluence binding to one ligand but not to the other, or improveactivity through one ligand while decreasing the activity through theother. Moreover, because the affinities of B7 molecules to their ligandsappear to be relatively low, it would also be desirable to findmutations that improve/alter the activities of the molecules. However,rational design does not enable predictions of useful mutations becauseof the complexity of the molecules.

The invention provides methods of overcoming these difficulties,enabling one to generate and identify functionally different B7molecules with altered relative capacities to induce T cell activation,differentiation, cytokine production, anergy and/or tolerance. Throughuse of the methods of the invention, one can find mutations in B7-1 andB7-2 that only influence binding to one ligand but not to the other, orthat improve activity through one ligand while decreasing the activitythrough the other. DNA shuffling is likely to be the most powerfulmethod in discovering new B7 variants with altered relative bindingcapacities to CD28 and CTLA-4. B7 variants which act through CD28 withimproved activity (and with decreased activity through CTLA-4) areexpected to have improved capacity to induce activation of T cells. Incontrast, B7 variants which bind and act through CTLA-4 with improvedactivity (and with decreased activity through CD28) are expected to bepotent negative regulators of T cell functions and to induce toleranceand anergy.

DNA shuffling or other recombination method is used to generate B7(e.g., B7-1/CD80 and B7-2/CD86) variants which have altered relativecapacity to act through CD28 and CTLA-4 when compared to wild-type B7molecules. In a preferred embodiment, the different forms of substrateused in the recombination reaction are B7 cDNAs from various species.Such cDNAs can be obtained by methods known to those of skill in theart, including RT-PCR.

Typically, genes encoding these variant B7 molecules are incorporatedinto genetic vaccine vectors encoding an antigen, so that one thevectors can be used to modify antigen-specific T cell responses. Vectorsthat harbor B7 genes that efficiently act through CD28 are useful ininducing, for example, protective immune responses, whereas vectors thatharbor genes encoding B7 genes that efficiently act through CTLA-4 areuseful in inducing, for example, tolerance and anergy of allergen- orautoantigen-specific T cells. In some situations, such as in tumor cellsor cells inducing autoimmune reactions, the antigen may already bepresent on the surface of the target cell, and the variant B7 moleculesmay be transfected in the absence of additional exogenous antigen gene.FIG. 11 illustrates a screening protocol that one can use to identifyB7-1 (CD80) and/or B7-2 (CD86) variants that have increased capacity toinduce T cell activation or anergy, and the application of this strategyis described in more detail in Example 1.

Several approaches for screening of the variants can be taken. Forexample, one can use a flow cytometry-based selection systems. Thelibrary of B7-1 and B7-2 molecules is transfected into cells thatnormally do not express these molecules (e.g., COS-7 cells or any cellline from a different species with limited or no cross-reactivity withman regarding B7 ligand binding). An internal marker gene can beincorporated in order to analyze the copy number per cell. SolubleCTLA-4 and CD28 molecules can be generated to or use in the flowcytometry experiments. Typically, these will be fused with the Fcportion of IgG molecule to improve the stability of the molecules and toenable easy staining by labeled anti-IgG mAbs, as described by van derMerwe et al. (J. Exp. Med. 185: 393, 1997). The cells transfected withthe library of B7 molecules are then stained with the soluble CTLA-4 andCD28 molecules. Cells demonstrating increased or decreased CTLA-4/CD28binding ratio will be sorted. The plasmids are then recovered and theshuffled B7 variant-encoding sequences identified. These selected B7variants can then be subjected to new rounds of shuffling and selection,and/or they can be further analyzed using functional assays as describedbelow.

The B7 variants can also be directly selected based on their functionalproperties. For in vivo studies, the B7 molecules can also be evolved tofunction on mouse cells. Bacterial colonies with plasmids with mutant B7molecules are picked and the plasmids are isolated. These plasmids arethen transfected into antigen presenting cells, such as dendritic cells,and the capacities of these mutants to activate T cells is analyzed. Oneof the advantages of this approach is that no assumptions on the bindingaffinities or specificities to the known ligands are made, and possiblynew activities through yet to be identified ligands can be found. Inaddition to dendritic cells, other cells that are relatively easy totransfect (e.g., U937 or COS-7) can be used in the screening, providedthat the “first T cell signal” is induced by, for example, anti-CD3monoclonal antibodies. T cell activation can be analyzed by methodsknown to those of skill in the art, including, for example, measuringproliferation, cytokine production, CTL activity or expression ofactivation antigens such as IL-2 receptor, CD69 or HLA-DR molecules.Usage of antigen-specific T cell clones, such as T cells specific forhouse dust mite antigen Der p I, will allow analysis of antigen-specificT cell activation (Yssel et al. (1992) J. Immunol. 148: 738-745).Mutants are identified that can enhance or inhibit T cell proliferationor enhance or inhibit CTL responses. Similarly variants that havealtered capacity to induce cytokine production or expression ofactivation antigens as measured by, for example, cytokine-specificELISAs or flow cytometry can be identified.

The B7 variants are useful in modulating immune responses in autoimmunediseases, allergy, cancer, infectious disease and vaccination. B7variants which act through CD28 with improved activity (and withdecreased activity through CTLA-4) will have improved capacity to induceactivation of T cells. In contrast, B7 variants which bind and actthrough CTLA-4 with improved activity (and with decreased activitythrough CD28) will be potent negative regulators of T cell functions andto induce tolerance and anergy. Thus, by incorporating genes encodingthese variant B7 molecules into genetic vaccine vectors encoding anantigen, it is possible to modify antigen-specific T cell responses.Vectors that harbor B7 genes that efficiently act through CD28 areuseful in inducing, for example, protective immune responses, whereasvectors that harbor genes encoding B7 genes that efficiently act throughCTLA-4 are useful in inducing, for example, tolerance and anergy ofallergen- or autoantigen-specific T cells. In some situations, such asin tumor cells or cells inducing autoimmune reactions, the antigen mayalready be present on the surface of the target cell, and the variant B7molecules may be transfected in the absence of additional exogenousantigen gene.

The methods of the invention are also useful for obtaining B7 variantsthat have increased effectiveness in directing either T_(H)1 or T_(H)2cell differentiation. Differential roles have been observed for B7-1 andB7-2 molecules in the regulation of T helper (T_(H)) celldifferentiation (Freeman et al. (1995) Immunity 2: 523; Kuchroo et al.(1995) Cell 80: 707). T_(H) cell differentiation can be measured byanalyzing the cytokine production profiles induced by each particularvariant. High levels of IL-4, IL-5 and/or IL-13 are an indication ofefficient T_(H)2 cell differentiation whereas high levels of IFN-γ orIL-2 production can be used as a marker of T_(H)1 cell differentiation.B7 variants with altered capacity to induce T_(H)1 or T_(H)2 celldifferentiation are useful, for example, in the treatment of allergic,malignant, autoimmune and infectious diseases and in vaccination.

Also provided by the invention are methods of obtaining B7 variants thathave enhanced capacity to induce IL-10 production by antigen-specific Tcells. Elevated production of IL-10 is a characteristic of regulatory Tcells, which can suppress proliferation of antigen-specific CD4′ T cells(Groux et al. (1997) Nature 389: 737). DNA shuffling is performed asdescribed above, after which recombinant nucleic acids encoding B7variants having enhanced capability of inducing IL-10 can be identifiedby, for example, ELISA or flow cytometry using intracytoplasmic cytokinestaining. The variants that induce high levels of IL-10 production areuseful in the treatment of allergic and autoimmune diseases.

D. Optimization of Transport and Presentation of Antigens

The invention also provides methods of obtaining genetic vaccines andaccessory molecules that can improve the transport and presentation ofantigenic peptides. A library of recombinant polynucleotides is createdand screened to identify those that encode molecules that have improvedproperties compared to the wild-type counterparts. The polynucleotidesthemselves can be used in genetic vaccines, or the gene products of thepolynucleotides can be utilized for therapeutic or prophylacticapplications.

1. Proteasomes

The class I peptides presented on major histocompatibility complexmolecules are generated by cellular proteasomes. Interferon-gamma canstimulate antigen presentation, and part of the mechanism of action ofinterferon may be due to induction of the proteasome beta-subunits LMP2and LMP7, which replace the homologous beta-subunits Y (delta) and X(epsilon). Such a replacement changes the peptide cleavage specificityof the proteasome and can enhance class I epitope immunogenicity. The Y(delta) and X (epsilon) subunits, as well as other recently discoveredproteasome subunits such as the MECL-1 homologue MC14, arecharacteristic of cells which are not specialized in antigenpresentation. Thus, the incorporation into cells by DNA transfer ofLMP2, LMP7, MECL-1 and/or other epitope presentation-specific andpotentially interferon-inducible subunits can enhance epitopepresentation. It is likely that the peptides generated by the proteasomecontaining the interferon-inducible subunits are transported to theendoplasmic reticulum by the TAP molecules.

The invention provides methods of obtaining proteasomes that exhibitincreased or decreased ability to specifically process MHC class Iepitopes. According to the methods, DNA shuffling is used to obtainevolved proteins that can either have new specificities which mightenhance the immunogenicity of some proteins and/or enhance the activityof the subunits once they are bound to the proteasome. Because thetransition from a non-specific proteasome to a class I epitope-specificproteasome can pass through several states (in which some but not all ofthe interferon-inducible subunits are associated with the proteasome),many different proteolytic specificities can potentially be achieved.Evolving the specific LMP-like subunits can therefore create newproteasome compositions which have enhanced functionality for thepresentation of epitopes.

The methods involve performing DNA shuffling using as substrates two ormore forms of polynucleotides which encode proteasome components, wherethe forms of polynucleotides differ in at least one nucleotide.Shuffling is performed as described herein, using polynucleotides thatencode any one or more of the various proteasome components, including,for example, LMP2, LMP7, MECL-1 and other individual proteasomecomponents that are specifically involved in class I epitopepresentation. Examples of suitable substrates are described in, e.g.,Stohwasser et al. (1997) Eur. J. Immunol. 27: 1182-1187 and Gaczynska etal: (1996) J. Biol. Chem. 271: 17275-17280. In a preferred embodiment,family shuffling is used, in which the different substrates areproteasome component-encoding polynucleotides from different species.

After the recombination reaction is completed, the resulting library ofrecombinant polynucleotides is screened to identify those which encodeproteasome components having the desired effect on class I epitopeproduction. For example, the recombinant polynucleotides can beintroduced into a genetic vaccine vector which also encodes a particularantigen of interest. The library of vectors can then be introduced intomammalian cells which are then screened to identify cells which exhibitincreased antigen-specific immunogenicity. Methods of analyzingproteasome activity are described in, for example, Groettrup et al.(1997) Proc. Nat'l. Acad. Sci. USA 94: 8970-8975 and Groettrup et al.(1997) Eur. J. Immunol. 26: 863-869.

Alternatively, one can use the methods of the invention to evolveproteins which bind strongly to the proteasome but have decreased or noactivity, thus antagonizing the proteasome activity and diminishing acells ability to present class I molecules. Such molecules can beapplied to gene therapy protocols in which it is desirable to lower theimmunogenicity of exogenous proteins expressed in the cells as a resultof the gene therapy, and which would otherwise be processed for class Ipresentation allowing the cell to be recognized by the immune system.Such high-affinity low-activity LMP-like subunits will demonstrateimmunosuppressive effects which are also of use in other therapeuticprotocols where cells expressing a non-self protein need to be protectedfrom an immune response.

The specificity of the proteasome and the TAP molecules (discussedbelow) may have co-evolved naturally. Thus it may be important that thetwo pathways of the class I processing system be functionally matched. Afurther aspect of the invention involves performing DNA shufflingsimultaneously on the two gene families followed by random combinationsof the two in order to discover appropriate matched proteolytic andtransport specificities.

2. Antigen Transport

The invention provides methods of improving transport of antigenicpeptides from the cytosolic compartment to the endoplasmic reticulum andthereby to the cell surface in the context of MHC class I molecules.Enhanced expression of antigenic peptides results in enhanced immuneresponse, particularly in improved activation of CD8⁺ cytotoxiclymphocytes. This is useful in the development of DNA vaccines and ingene therapy.

In one embodiment, the invention involves evolving TAP-genes(transporters associated with antigen processing) to obtain genes thatexhibit improved antigen presentation. TAP genes are members ofATP-binding cassette family of membrane translocators. These proteinstransport antigenic peptides to MHC class I molecules and are involvedin the expression and stability of MHC class I molecules on the cellsurface. Two TAP genes, TAP 1 and TAP2, have been cloned to date (Powiset al. (1996) Proc. Nat'l. Acad. Sci. USA 89: 1463-1467; Koopman et al.(1997) Curr. Opin. Immunol. 9: 80-88; Monaco (1995) J Leukocyte Biol.57: 543-57). TAP1 and TAP2 form a heterodimer and these genes arerequired for transport of peptides into the endoplasmic reticulum, wherethey bind to MHC class I molecules. The essential role of TAP geneproducts in presentation of antigenic peptides was demonstrated in micewith disrupted TAP genes. TAP1-deficient mice have drastically reducedlevels of surface expression of MHC class I, and positive selection ofCD8⁺ T cells in the thymus is strongly reduced. Therefore, the number ofCD8⁺ T lymphocytes in the periphery of TAP-deficient mice is extremelylow. Transfection of TAP genes back into these cells restores the levelof MHC class I expression.

TAP genes are a good target for gene shuffling because of naturalpolymorphism and because these genes of several mammalian species havebeen cloned and sequenced, including human (Beck et al. (1992) J. Mol.Biol. 228: 433-441; Genbank Accession No. Y13582; Powis et al., supra.),gorilla TAP1 (Laud et al. (1996) Human Immunol. 50: 91-102), mouse(Reiser et al. (1988) Proc. Nat'l. Acad. Sci. USA 85: 2255-2259;Marusina et al. (1997) J. Immunol. 158: 5251-5256, TAP1: GenbankAccession Nos. U60018, U60019, U60020, U60021, U60022, andL76468-L67470; TAP2: Genbank Accession Nos. U60087, U60088, U6089,U60090, U60091 and U60092), hamster (TAP1, Genbank Accession Nos.AF001154 and AF00157; TAP2, Genbank Accession Nos. AF001156 andAF00155). Furthermore, it has been shown that point mutations in TAPgenes may result in altered peptide specificity and peptidepresentation. Also, functional differences in TAP genes derived fromdifferent species have been observed. For example, human TAP and rat TAPcontaining the rTAP2a allele are rather promiscuous, whereas mouse TAPis restrictive and select against peptides with C-terminal smallpolar/hydrophobic or positively charged amino acids. The basis for thisselectivity is unknown.

The methods of the invention involve performing DNA shuffling of TAP1and TAP2 genes using as substrates at least two forms of TAP1 and/orTAP2 polynucleotide sequences which differ in at least one nucleotideposition. In a preferred embodiment, TAP sequences derived from severalmammalian species are used as the substrates for shuffling. Naturalpolymorphism of the genes can provide additional diversity of substrate.If desired, optimized TAP genes obtained from one round of shuffling andscreening can be subjected to additional shuffling/screening rounds toobtain further optimized TAP-encoding polynucleotides.

To identify optimized TAP-encoding polynucleotides from a library ofrecombinant TAP genes, the genes can be expressed on the same plasmid asa target antigen of interest. If this step is limiting the extent ofantigen presentation, then enhanced presentation to CD8⁺ CTL willresult. Mutants of TAPs may act selectively to increase expression of aparticular antigen peptide fragment for which levels of expression areotherwise limiting, or to cause transport of a peptide that wouldnormally never be transferred into the RER and made available to bind toMHC Class I.

When used in the context of gene therapy vectors in cancer treatment,evolved TAP genes provide a means to enhance expression of MHC class Imolecules on tumor cells and obtain efficient presentation of antigenictumor-specific peptides. Thus, vectors that contain the evolved TAPgenes can induce potent immune responses against the malignant cells.Shuffled TAP genes can be transfected into malignant cell lines thatexpress low levels of MHC class I molecules using retroviral vectors orelectroporation. Transfection efficiency can be monitored using markergenes, such as green fluorescent protein, encoded by the same vector asthe TAP genes. Cells expressing equal levels of green fluorescentprotein but the highest levels of MHC class I molecules, as a marker ofefficient TAP genes, are then sorted using flow cytometry, and theevolved TAP genes are then recovered from these cells by, for example,PCR or by recovering the entire vectors. These sequences can thensubjected into new rounds of shuffling, selection and recovery, iffurther optimization is desired.

Molecular evolution of TAP genes can be combined with simultaneousevolution of the desired antigen. Simultaneous evolution of the desiredantigen can further improve the efficacy of presentation of antigenicpeptides following DNA vaccination. The antigen can be evolved, usinggene shuffling, to contain structures that allow optimal presentation ofdesired antigenic peptides when optimal TAP genes are expressed. TAPgenes that are optimal for presentation of antigenic peptides of onegiven antigen may be different from TAP genes that are optimal forpresentation of antigenic peptide of another antigen. Gene shufflingtechnique is ideal, and perhaps the only, method to solve this type ofproblems. Efficient presentation of desired antigenic peptides can beanalyzed using specific cytotoxic T lymphocytes, for example; bymeasuring the cytokine production or CTL activity of the T lymphocytesusing methods known to those of skill in the art.

3. Cytotoxic T-Cell Inducing Sequences and Immunogenic Agonist Sequences

Certain proteins are better able than others to carry MHC class Iepitopes because they are more readily used by the cellular machineryinvolved in the necessary processing for class I epitope presentation.The invention provides methods of identifying expressed polypeptidesthat are particularly efficient in traversing the various biosyntheticand degradative steps leading to class I epitope presentation and theuse of these polypeptides to enhance presentation of CTL epitopes fromother proteins.

In one embodiment, the invention provides Cytotoxic T-cell InducingSequences (CTIS), which can be used to carry heterologous class Iepitopes for the purpose of vaccinating against the pathogen from whichthe heterologous epitopes are derived. One example of a CTIS is obtainedfrom the hepatitis B surface antigen (HBsAg), which has been shown to bean effective carrier for its own CTL epitopes when delivered as aprotein under certain conditions. DNA immunization with plasmidsexpressing the HBsAg also induces high levels of CTL activity. Theinvention provides a shorter, truncated fragment of the HBsAgpolypeptide which functions very efficiently in inducing CTL activity,and attains CTL induction levels that are higher than with the HBsAgprotein or with the plasmids encoding the full-length HBsAg polypeptide.Synthesis of a CTIS derived from HBsAg is described in Example 3; and adiagram of a CTIS is shown in FIG. 1.

The ER localization of the truncated polypeptide may be important inachieving suitable proteolytic liberation of the peptide(s) containingthe CTL epitopes (see Cresswell & Hughes (1997) Curr. Biol. 7:R552-R555; Craiu et al. (1997) Proc. Nat'l. Acad. Sci. USA 94:10850-10855). The preS2 region and the transmembrane region provideT-helper epitopes which may be important for the induction of a strongcytotoxic immune response. Because the truncated CTIS polypeptide has asimple structure (see FIG. 1), it is possible to attach one or moreheterologous class I epitope sequences to the C-terminal end of thepolypeptide without having to maintain any specific proteinconformation. Such sequences are then available to the class I epitopeprocessing mechanisms. The size of the polypeptide is not subject to thenormal constraints of the native HBsAg structure. Therefore the lengthof the heterologous sequence and thus the number of included CTLepitopes is flexible. This is shown schematically in FIG. 2. The abilityto include a long sequence containing either multiple and distinct classI sequences, or alternatively different variations of a single CTLsequence, allows DNA shuffling methodology to be applied.

The invention also provides methods of obtaining Immunogenic AgonistSequences (IAS) which induce CTLs capable of specific lysis of cellsexpressing the natural epitope sequence. In some cases, the reactivityis greater than if the CTL response is induced by the natural epitope(see, Example 3 and FIG. 3). Such IAS-induced CTL may be drawn from aT-cell repertoire different from that induced by the natural sequence.In this way, poor responsiveness to a given epitope can be overcome byrecruiting T cells from a larger pool. In order to discover such IAS,the amino acid at each position of a CTL-inducing peptide (excludingperhaps the positions of the so-called anchor residues) can be variedover the range of the 19 amino acids not normally present at theposition. DNA shuffling methodology can be used to scan a large range ofsequence possibilities.

A synthetic gene segment containing multiple copies of the originalepitope sequence can be prepared such that each copy possesses a smallnumber of nucleotide changes. The gene segment can be shuffled to createa diverse range of CTL epitope sequences, some of which should functionas IAS. This process is illustrated in FIG. 4.

In practice, oligonucleotides are typically constructed in accordancewith the above design and polymerized enzymatically to form thesynthetic gene segment of the concatenated epitopes. Restriction sitescan be incorporated into a fraction of the oligonucleotides to allow forcleavage and selection of given size ranges of the concatenatedepitopes, most of which will have different sequences and thus will bepotential IAS. The epitope-containing gene segment can be joined byappropriate cloning methods to a CTIS, such as that of HBsAg. Theresulting plasmid constructions can be used for DNA-based immunizationand CTL induction.

E. Genetic Vaccine Pharmaceutical Compositions and Methods ofAdministration

The improved immunomodulatory polynucleotides and polypeptides of theinvention are useful for treating and/or preventing the various diseasesand conditions with which the respective antigens are associated. Forexample, genetic vaccines that employ the reagents obtained according tothe methods of the invention are useful in both prophylaxis and therapyof infectious diseases, including those caused by any bacterial, fungal,viral, or other pathogens of mammals. The reagents obtained using theinvention can also be used for treatment of autoimmune diseasesincluding, for example, rheumatoid arthritis, SLE, diabetes mellitus,myasthenia gravis, reactive arthritis, ankylosing spondylitis, andmultiple sclerosis. These and other inflammatory conditions, includingIBD, psoriasis, pancreatitis, and various immunodeficiencies, can betreated using genetic vaccines that include vectors and other componentsobtained using the methods of the invention. Genetic vaccine vectors andother reagents obtained using the methods of the invention can be usedto treat allergies and asthma. Moreover, the use of genetic vaccineshave great promise for the treatment of cancer and prevention ofmetastasis. By inducing an immune response against cancerous cells, thebody's immune system can be enlisted to reduce or eliminate cancer.

In presently preferred embodiments, the reagents obtained using theinvention are used in conjunction with a genetic vaccine vector. Thechoice of vector and components can also be optimized for the particularpurpose of treating allergy or other conditions. For example, an antigenassociated with treating a particular condition can be optimized usingrecombination and selection methods analogous to those described herein.Such methods, and antigens appropriate for various conditions, aredescribed in copending, commonly assigned U.S. patent application Ser.No. ______, entitled “Antigen Library Immunization,” which was filed onFeb. 10, 1999 as TTC Attorney Docket No. 18097-028710US. Thepolynucleotide that encodes the recombinant antigenic polypeptide can beplaced under the control of a promoter, e.g., a high activity ortissue-specific promoter. The promoter used to express the antigenicpolypeptide can itself be optimized using recombination and selectionmethods analogous to those described herein, as described inInternational Application No. PCT/US97/17300 (International PublicationNo. WO 98/13487). The reagents obtained using the methods of theinvention can also be used in conjunction with multicomponent geneticvaccines, which are capable of tailoring an immune response as is mostappropriate to achieve a desired effect (see, e.g., copending, commonlyassigned U.S. patent application Ser. No. ______, entitled “GeneticVaccine Vector Engineering,” filed on Feb. 10, 1999 as TTC AttorneyDocket No. 18097-030100US). It is sometimes advantageous to employ agenetic vaccine that is targeted for a particular target cell type(e.g., an antigen presenting cell or an antigen processing cell);suitable targeting methods are described in copending, commonly assignedU.S. patent application Ser. No. ______, entitled “Targeting of GeneticVaccine Vectors,” filed on Feb. 10, 1999 as TTC Attorney Docket No.18097-030200US.

Genetic vaccine vectors that include the optimized recombinantpolynucleotides obtained as described herein can be delivered to amammal (including humans) to induce a therapeutic or prophylactic immuneresponse. Vaccine delivery vehicles can be delivered in vivo byadministration to an individual patient, typically by systemicadministration (e.g., intravenous, intraperitoneal, intramuscular,subdermal, intracranial, anal, vaginal, oral, buccal route or they canbe inhaled) or they can be administered by topical application.Alternatively, vectors can be delivered to cells ex vivo, such as cellsexplanted from an individual patient (e.g., lymphocytes, bone marrowaspirates, tissue biopsy) or universal donor hematopoietic stem cells,followed by reimplantation of the cells into a patient, usually afterselection for cells which have incorporated the vector.

A large number of delivery methods are well known to those of skill inthe art. Such methods include, for example liposome-based gene delivery(Debs and Zhu (1993) WO 93/24640; Mannino and Gould-Fogerite (1988)BioTechniques 6(7): 682-691; Rose U.S. Pat. No. 5,279,833; Brigham(1991) WO 91/06309; and Felgner et al. (1987) Proc. Natl. Acad. Sci. USA84: 7413-7414), as well as use of viral vectors (e.g., adenoviral (see,e.g., Berns et al. (1995) Ann. NY Acad. Sci. 772: 95-104; Ali et al.(1994) Gene Ther. 1: 367-384; and Haddada et al. (1995) Curr. Top.Microbiol. Immunol. 199 (Pt 3); 297-306 for review), papillomaviral,retroviral (see, e.g., Buchscher et al. (1992) J. Virol. 66(5)2731-2739; Johann et al. (1992) J. Virol. 66 (5):1635-1640 (1992);Sommerfelt et al., (1990) Virol. 176:58-59; Wilson et al. (1989) J.Virol. 63:2374-2378; Miller et al., J. Virol. 65:2220-2224 (1991);Wong-Staal et al., PCT/US94/05700, and Rosenburg and Fauci (1993) inFundamental Immunology, Third Edition Paul (ed) Raven Press, Ltd., NewYork and the references therein, and Yu et al., Gene Therapy (1994)supra.), and adeno-associated viral vectors (see, West et al. (1987)Virology 160:38-47; Carter et al. (1989) U.S. Pat. No. 4,797,368; Carteret al. WO 93/24641 (1993); Kotin (1994) Human Gene Therapy 5:793-801;Muzyczka (1994) J. Clin. Invst. 94:1351 and Samulski (supra) for anoverview of AAV vectors; see also, Lebkowski, U.S. Pat. No. 5,173,414;Tratschin et al. (1985) Mol. Cell. Biol. 5(11):3251-3260; Tratschin, etal. (1984) Mol. Cell. Biol., 4:2072-2081; Hermonat and Muzyczka (1984)Proc. Natl. Acad. Sci. USA, 81:6466-6470; McLaughlin et al. (1988) andSamulski et al. (1989) J. Virol., 63:03822-3828), and the like.

“Naked” DNA and/or RNA that comprises a genetic vaccine can beintroduced directly into a tissue, such as muscle. See, e.g., U.S. Pat.No. 5,580,859. Other methods such as “biolistic” or particle-mediatedtransformation (see, e.g., Sanford et al., U.S. Pat. No. 4,945,050; U.S.Pat. No. 5,036,006) are also suitable for introduction of geneticvaccines into cells of a mammal according to the invention. Thesemethods are useful not only for in vivo introduction of DNA into amammal, but also for ex vivo modification of cells for reintroductioninto a mammal. As for other methods of delivering genetic vaccines, ifnecessary, vaccine administration is repeated in order to maintain thedesired level of immunomodulation.

Genetic vaccine vectors (e.g., adenoviruses, liposomes,papillomaviruses, retroviruses, etc.) can be administered directly tothe mammal for transduction of cells in vivo. The genetic vaccinesobtained using the methods of the invention can be formulated aspharmaceutical compositions for administration in any suitable manner,including parenteral (e.g., subcutaneous, intramuscular, intradermal, orintravenous), topical, oral, rectal, intrathecal, buccal (e.g.,sublingual), or local administration, such as by aerosol ortransdermally, for prophylactic and/or therapeutic treatment.Pretreatment of skin, for example, by use of hair-removing agents, maybe useful in transdermal delivery. Suitable methods of administeringsuch packaged nucleic acids are available and well known to those ofskill in the art, and, although more than one route can be used toadminister a particular composition, a particular route can oftenprovide a more immediate and more effective reaction than another route.

Pharmaceutically acceptable carriers are determined in part by theparticular composition being administered, as well as by the particularmethod used to administer the composition. Accordingly, there is a widevariety of suitable formulations of pharmaceutical compositions of thepresent invention. A variety of aqueous carriers can be used, e.g.,buffered saline and the like. These solutions are sterile and generallyfree of undesirable matter. These compositions may be sterilized byconventional, well known sterilization techniques. The compositions maycontain pharmaceutically acceptable auxiliary substances as required toapproximate physiological conditions such as pH adjusting and bufferingagents, toxicity adjusting agents and the like, for example, sodiumacetate, sodium chloride, potassium chloride, calcium chloride, sodiumlactate and the like. The concentration of genetic vaccine vector inthese formulations can vary widely, and will be selected primarily basedon fluid volumes, viscosities, body weight and the like in accordancewith the particular mode of administration selected and the patient'sneeds.

Formulations suitable for oral administration can consist of (a) liquidsolutions, such as an effective amount of the packaged nucleic acidsuspended in diluents, such as water, saline or PEG 400; (b) capsules,sachets or tablets, each containing a predetermined amount of the activeingredient, as liquids, solids, granules or gelatin; (c) suspensions inan appropriate liquid; and (d) suitable emulsions. Tablet forms caninclude one or more of lactose, sucrose, mannitol, sorbitol, calciumphosphates, corn starch, potato starch, tragacanth, microcrystallinecellulose, acacia, gelatin, colloidal silicon dioxide, croscarmellosesodium, talc, magnesium stearate, stearic acid, and other excipients,colorants, fillers, binders, diluents, buffering agents, moisteningagents, preservatives, flavoring agents, dyes, disintegrating agents,and pharmaceutically compatible carriers. Lozenge forms can comprise theactive ingredient in a flavor, usually sucrose and acacia or tragacanth,as well as pastilles comprising the active ingredient in an inert base,such as gelatin and glycerin or sucrose and acacia emulsions, gels, andthe like containing, in addition to the active ingredient, carriersknown in the art. It is recognized that the genetic vaccines, whenadministered orally, must be protected from digestion. This is typicallyaccomplished either by complexing the vaccine vector with a compositionto render it resistant to acidic and enzymatic hydrolysis or bypackaging the vector in an appropriately resistant carrier such as aliposome. Means of protecting vectors from digestion are well known inthe art. The pharmaceutical compositions can be encapsulated, e.g., inliposomes, or in a formulation that provides for slow release of theactive ingredient.

The packaged nucleic acids, alone or in combination with other suitablecomponents, can be made into aerosol formulations (e.g., they can be“nebulized”) to be administered via inhalation. Aerosol formulations canbe placed into pressurized acceptable propellants, such asdichlorodifluoromethane, propane, nitrogen, and the like.

Suitable formulations for rectal administration include, for example,suppositories, which consist of the packaged nucleic acid with asuppository base. Suitable suppository bases include natural orsynthetic triglycerides or paraffin hydrocarbons. In addition, it isalso possible to use gelatin rectal capsules which consist of acombination of the packaged nucleic acid with a base, including, forexample, liquid triglycerides, polyethylene glycols, and paraffinhydrocarbons.

Formulations suitable for parenteral administration, such as, forexample, by intraarticular (in the joints), intravenous, intramuscular,intradermal, intraperitoneal, and subcutaneous routes, include aqueousand non-aqueous, isotonic sterile injection solutions, which can containantioxidants, buffers, bacteriostats, and solutes that render theformulation isotonic with the blood of the intended recipient, andaqueous and non-aqueous sterile suspensions that can include suspendingagents, solubilizers, thickening agents, stabilizers, and preservatives.In the practice of this invention, compositions can be administered, forexample, by intravenous infusion, orally, topically, intraperitoneally,intravesically or intrathecally. Parenteral administration andintravenous administration are the preferred methods of administration.The formulations of packaged nucleic acid can be presented in unit-doseor multi-dose sealed containers, such as ampoules and vials.

Injection solutions and suspensions can be prepared from sterilepowders, granules, and tablets of the kind previously described. Cellstransduced by the packaged nucleic acid can also be administeredintravenously or parenterally.

The dose administered to a patient, in the context of the presentinvention should be sufficient to effect a beneficial therapeuticresponse in the patient over time. The dose will be determined by theefficacy of the particular vector employed and the condition of thepatient, as well as the body weight or vascular surface area of thepatient to be treated. The size of the dose also will be determined bythe existence, nature, and extent of any adverse side-effects thataccompany the administration of a particular vector, or transduced celltype in a particular patient.

In determining the effective amount of the vector to be administered inthe treatment or prophylaxis of an infection or other condition, thephysician evaluates vector toxicities, progression of the disease, andthe production of anti-vector antibodies, if any. In general, the doseequivalent of a naked nucleic acid from a vector is from about 1 μg to 1mg for a typical 70 kilogram patient, and doses of vectors used todeliver the nucleic acid are calculated to yield an equivalent amount oftherapeutic nucleic acid. Administration can be accomplished via singleor divided doses.

In therapeutic applications, compositions are administered to a patientsuffering from a disease (e.g., an infectious disease or autoimmunedisorder) in an amount sufficient to cure or at least partially arrestthe disease and its complications. An amount adequate to accomplish thisis defined as a “therapeutically effective dose.” Amounts effective forthis use will depend upon the severity of the disease and the generalstate of the patient's health. Single or multiple administrations of thecompositions may be administered depending on the dosage and frequencyas required and tolerated by the patient. In any event, the compositionshould provide a sufficient quantity of the proteins of this inventionto effectively treat the patient.

In prophylactic applications, compositions are administered to a humanor other mammal to induce an immune response that can help protectagainst the establishment of an infectious disease or other condition.

The toxicity and therapeutic efficacy of the genetic vaccine vectorsprovided by the invention are determined using standard pharmaceuticalprocedures in cell cultures or experimental animals. One can determinethe LD₅₀ (the dose lethal to 50% of the population) and the ED₅₀ (thedose therapeutically effective in 50% of the population) usingprocedures presented herein and those otherwise known to those of skillin the art.

A typical pharmaceutical composition for intravenous administrationwould be about 0.1 to 10 mg per patient per day. Dosages from 0.1 up toabout 100 mg per patient per day may be used, particularly when the drugis administered to a secluded site and not into the blood stream, suchas into a body cavity or into a lumen of an organ. Substantially higherdosages are possible in topical administration. Actual methods forpreparing parenterally administrable compositions will be known orapparent to those skilled in the art and are described in more detail insuch publications as Remington's Pharmaceutical Science, 15th ed., MackPublishing Company, Easton, Pa. (1980).

The multivalent antigenic polypeptides of the invention, and geneticvaccines that express the polypeptides, can be packaged in packs,dispenser devices, and kits for administering genetic vaccines to amammal. For example, packs or dispenser devices that contain one or moreunit dosage forms are provided. Typically, instructions foradministration of the compounds will be provided with the packaging,along with a suitable indication on the label that the compound issuitable for treatment of an indicated condition.

For example, the label may state that the active compound within thepackaging is useful for treating a particular infectious disease,autoimmune disorder, tumor, or for preventing or treating other diseasesor conditions that are mediated by, or potentially susceptible to, amammalian immune response.

EXAMPLES

The following examples are offered to illustrate, but not to limit thepresent invention.

Example 1 Altered Ligand Specificity of B7-1 (CD80) and/or B7-2 (CD86)by DNA Shuffling

This Example describes the use of the DNA shuffling methods of theinvention to obtain B7-1 and B7-2 polypeptides that have alteredbiological activities.

DNA Shuffling

DNA shuffling is used to generate a library of B7 (B7-1/CD80 andB7-2/CD86) variants that have altered relative capacity to act throughCD28 and CTLA-4 when compared to wild-type B7 molecules. Typically, B7cDNAs from various species are generated by RT-PCR, and these sequencesare shuffled using family DNA shuffling. Alignments of human, rhesusmonkey and rabbit B7-1 nucleotide sequences are shown in FIG. 15,demonstrating that family DNA shuffling is a feasible approach whenevolving B7 molecules.

Screening of B7 Variants

The library is then screened to identify those variants that are usefulin modulating immune responses in autoimmune diseases, allergy, cancer,infectious disease and vaccination. Any of several approaches forscreening of the variants can be used:

A. Flow Cytometry-Based Selection System.

The library of B7-1 and B7-2 molecules is transfected into cells thatnormally do not express these molecules (e.g., COS-7 cells or any cellline from different species with limited or no cross-reactivity with manregarding B7 ligand binding). An internal marker gene can beincorporated in order to analyze the copy number per cell.

Soluble CTLA-4 and CD28 molecules are generated to facilitate the flowcytometry experiments. Typically, these soluble polypeptides are fusedwith the Fc portion of IgG molecule to improve the stability of themolecules and to enable easy staining by labeled anti-IgG monoclonalantibodies, as described by van der Merwe et al. ((1997) J. Exp. Med.185: 393). The cells transfected with the library of B7 molecules arethen stained with the soluble CTLA-4 and CD28 molecules. Cellsdemonstrating increased or decreased CTLA-4/CD28 binding ratio aresorted. The plasmids are then recovered and the shuffled sequencesidentified. These selected B7 variants can then be subjected to newrounds of shuffling and selection, and can be further analyzed usingfunctional assays as described below.

B. Selection Based on Functional Properties.

Bacterial colonies that contain plasmids that include mutant B7molecules are picked and the plasmids are isolated. These plasmids arethen transfected into antigen presenting cells, such as dendritic cells,and the capacities of these mutants to activate T cells is analyzed. Oneof the advantages of this approach is that no assumptions are made as tothe binding affinities or specificities to the known ligands, andpossibly new activities through yet to be identified ligands can befound.

T cell activation can be analyzed by measuring proliferation, cytokineproduction, CTL activity or expression of activation antigens such asIL-2 receptor, CD69 or HLA-DR molecules. Usage of antigen-specific Tcell clones, such as T cells specific for house dust mite antigen Der pI, allows analysis of antigen-specific T cell activation. Mutants areidentified that can enhance or inhibit T cell proliferation or enhanceor inhibit CTL responses. Similarly variants that have altered capacityto induce cytokine production or expression of activation antigens asmeasured by, for example, cytokine-specific ELISAs or flow cytometry canbe selected. Results obtained using a proliferation-based assay is shownare shown in FIG. 13.

C. Ability to Direct Either T_(H)1 or T_(H)2 Cell Differentiation.

Because differential roles for B7-1 and B7-2 molecules in the regulationof T helper cell differentiation have been identified (Freeman et al.(1995) Immunity 2: 523; Kuchroo et al. (1995) Cell 80: 707), one canscreen for B7 variants that are the most effective in directing eitherT_(H)1 or T_(H)2 cell differentiation. T_(H) cell differentiation can bemeasured by analyzing the cytokine production profiles induced by eachparticular variant. High levels of IL-4, IL-5 and/or IL-13 are anindication of efficient T_(H)2 cell differentiation whereas high levelsof IFN-γ or IL-2 production can be used as a marker of T_(H)1 celldifferentiation. B7 variants that altered capacity to induce T_(H)1 orT_(H)2 cell differentiation are likely to be useful in the treatment ofallergic, malignant, autoimmune and infectious diseases and invaccination.

D. Enhanced IL-10 Production.

Elevated production of IL-10 is a characteristic of regulatory T cells,which can suppress proliferation of antigen-specific CD4⁺ T cells (Grouxet al. (1997) Nature 389: 737). Therefore, B7 variants can be screenedto identify those that have enhanced capacity to induce IL-10 productionby antigen-specific T cells. IL-10 production can be measured, forexample, by ELISA or flow cytometry using intracytoplasmic cytokinestainings. The variants that induce high levels of IL-10 production areuseful in the treatment of allergic and autoimmune diseases.

Example 2 Evolution Of Cytokines for Improved Specific Activity and/orImproved Expression Levels

This example describes a method to evolve a cytokine for improvedspecific activity and/or improved expression levels when the geneticvaccine is transfected into mammalian cells. IL-12 is the most potentcytokine directing T_(H)1 responses, and it improves the efficacy ofgenetic vaccinations. Evolved IL-12 molecules are useful as componentsof genetic vaccines. IL-12 is a heterodimeric cytokine composed of a 35kD light chain (p35) and a 40 kD heavy chain (p40) (Kobayashi et al.(1989) J. Exp. Med. 170: 827; Stern et al. (1990) Proc. Nat'l. Acad.Sci. USA 87: 6808). Recently Lieschke et al. (Nature Biotechnol. (1997)15: 35) demonstrated that a fusion between p35 and p40 genes results ina single gene that has activity comparable to that of the two genesexpressed separately. Accordingly, an IL-12 gene is shuffled as oneentity that encodes both subunits, which is beneficial in designing theshuffling protocol. The subunits of IL-12 can also be expressedseparately in the same expression vector, or the subunits can beexpressed separately and screened using cotransfections of the twovectors, providing additional shuffling strategies.

IL-12 plays-several roles in the regulation of allergic responses. Forexample, IL-12 induces T_(H)1 cell differentiation and downregulates theT_(H)2 response. IL-12 inhibits IgE synthesis both in vivo and in vitro,and also induces IFN-γ production. Accordingly, it is desirable toobtain an optimized IL-12 that better able to carry out these functionsupon administration to a mammal.

Cytokine genes, including IL-12 genes, from humans and nonhuman primatesare generally 93-99% homologous (Villinger et al. (1995) J. Immunol.0.155:3946-3954), providing a good starting point for family shuffling.A library of shuffled IL-12 genes was obtained by shuffling p35 and p40subunits derived from human, rhesus monkey, cat, dog, cow, pig, andgoat, and incorporated into vectors and the supernatants of thesetransfectants are analyzed for biological activity as shown in FIG. 6.Because of its T cell growth promoting activities, it is possible to usenormal human peripheral blood T cells in the selection of the mostactive IL-12 genes, enabling directly to select IL-12 mutants with themost potent activities on human T cells.

As shown in FIG. 7, a functional screening assay has been successfullyestablished. In this assay, COS-7 cells were first transfected withvectors encoding IL-12 subunits. Forty-eight hours after transfection,the capacity of these culture supernatants to induce proliferation ofactivated human peripheral blood T cells was studied. FIG. 8 indicatesthe consistency of the level of T cell proliferation induced in thisassay, indicating that the assay can be used to distinguish theactivities between supernatants that have different capacities to induceT cell activation. In other words, the assay provides means to screenfor improved IL-12-like activities in culture supernatants oftransfected cells. A vector with an optimized IL-12-encodingpolynucleotide was tested for ability to induce human T cell activation.Results, shown in FIG. 9, show that the shuffled IL-12 has ansignificantly increased ability to induce T cell activation compared towild-type IL-12.

FIG. 6 illustrates a general strategy for screening of evolved cytokinegenes. The specific example is given for IL-12 but similar approachapplies to all cytokines when using cell types sensitive for eachcytokine. For example, GM-CSF can be evolved by the same approach byusing the GM-CSF sensitive cell line TF-1 in the screening. In addition,although in this example the vectors are transfected into CHO cells, anymammalian cell that can be transfected in vitro can be used as hostcells. In addition to CHO cell, other good host cells include cell linesWI-26, COS-1, COS-7, 293, U937 and freshly isolated human antigenpresenting cells, such as monocytes, B cells and dendritic cells.

Example 3 Cytotoxic T-Cell Inducing Sequences Derived from Hepatitis BSurface Antigen and Strongly Immunogenic Agonistic T Cell Epitopes

This Example describes the preparation of a polypeptide sequence capableof efficient presentation of T cell epitopes and a strategy for theapplication of DNA shuffling to discover strongly immunogenic agonisticT cell epitopes.

The HBsAg polypeptide (PreS2 plus S regions) was truncated by theintroduction of a stop codon at amino acid position 103 (counting fromthe beginning of the PreS2 initiator methionine), transforming acysteine codon TGT into the Stop codon TGA. The amino acid sequence ofthe truncated protein was therefore:MQWNSTTFHQTLQDPRVRGLYFPAGGSSSGTVNPVLTTASPLSSIFSRIGDPALNMENITSGFLGPLLVLQAGFFLLTRILTIPQSLDSWWTSLNFLGGTTV* where the standardsingle-letter code for amino acids is used. The methionine residues atthe start of the PreS2 and S regions are underlined, and the mouseL^(d)-restricted CTL epitope is double-underlined; the asterisk (*)represents the artificially introduced Stop codon.

A likely structure for this truncated polypeptide is shown in FIG. 1.During protein biosynthesis, the N-terminal region of the HBsAgpolypeptide is transported through the membrane of the endoplasmicreticulum (ER). The first part of the S region is a transmembranestructure which locks the polypeptide into the ER. The remainingC-terminal region of the polypeptide is located in the cytoplasmiccompartment where it can be accessed by the epitope processing mechanismof the cell. This structure forms what is referred to as the CytotoxicT-cell Inducing Sequence (CTIS).

A CTIS is preferably used in conjunction with an immunogenic agonisticsequence (IAS). As an example of an IAS, each position of a 12 aminoacid L^(d)-restricted class I epitope from HBsAg was replaced with analanine-encoding codon in the DNA sequence of the epitope. The resultsdemonstrate that, in some cases, the reactivity is greater than if theCTL response is induced by the natural epitope (FIG. 3).

Example 4 Treatment of Obesity, Anorexia, and Cachexia Using OptimizedImmunomodulatory Molecules

Optimized immunomodulatory molecules that are obtained using the methodsof the invention find use in a wide variety of applications, in additionto use in vaccination. For example, there is increasing evidence thatcertain forms of obesity are associated with dysfunction of the immunesystem, and that molecules which regulate immune responses, e.g.cytokines, can induce or inhibit obesity. The invention provides methodsof optimizing immune regulatory molecules for the treatment of obesity,anorexia and cachexia.

Leptin and ciliary neurotrophic factor (CNTF) are examples of cytokinesthat have been shown to play a role in the development of obesity.Congenital leptin deficiency results in severe early-onset obesity inhuman (Montague et al. (1997) Nature 387: 903-908), and CNTF has beenshown to correct obesity and diabetes associated with leptin deficiencyand resistance (Gloaguen et al. (1997) Proc. Nat'l. Acad. Sci. USA 94:6456-6461). Antagonists of CNTF and/or leptin may be useful in thetreatment of anorexia and/or cachexia.

The methods of the invention are used to generate leptin and/or CNTFmolecules that have improved specific activity. The methods are alsouseful for obtaining improved cytokines that exhibit reducedimmunogenicity in vivo; immunogenicity is a particular concern for CNTF,because the wild-type CNTF is highly antigenic, which results in theproduction of high levels of anti-CNTF antibodies when administered to ahuman. Improved cytokine molecules prepared using the methods of theinvention are administered as polypeptides, or the shuffled nucleicacids that encode improved leptin and/or CNTF polypeptides are used ingenetic vaccine vectors. The invention also provides methods ofgenerating vectors that induce production of increased levels of leptinand/or CNTF. The methods of the invention can also be used to obtainreagents that are useful for the treatment of anorexia, cachexia, andrelated disorders. In this embodiment, antagonists of leptin and/or CNTFare evolved using the DNA shuffling methods. For example, a leptinreceptor can be evolved to obtain a soluble form that has an enhancedaffinity for leptin. The receptor for leptin in mice is found in thehypothalamus (Mercer et al. (1996) FEBS Lett. 387: 113), a region knownto be involved in maintenance of energy balance, and in the choroidplexus and leptomeninges, which form part of the blood/brain barrier.

It is understood that the examples and embodiments described herein arefor illustrative purposes only and that various modifications or changesin light thereof will be suggested to persons skilled in the art and areto be included within the spirit and purview of this application andscope of the appended claims. All publications, patents, and patentapplications cited herein are hereby incorporated by reference for allpurposes.

1. A method for obtaining a polynucleotide that has a modulatory effecton an immune response, or encodes a polypeptide that has a modulatoryeffect on an immune response, that is induced by a genetic vaccinevector, the method comprising: creating a library of recombinantpolynucleotides; and screening the library to identify an optimizedrecombinant polynucleotide that has, or encodes a polypeptide that has,a modulatory effect on an immune response induced by a genetic vaccinevector; wherein the optimized recombinant polynucleotide or thepolypeptide encoded by the recombinant polynucleotide exhibits anenhanced ability to modulate an immune response compared to anon-recombinant polynucleotide from which the library was created. 2.The method of claim 1, wherein the optimized recombinant polynucleotideis incorporated into a genetic vaccine vector.
 3. The method of claim 1,wherein the optimized recombinant polynucleotide, or a polypeptideencoded by the optimized recombinant polynucleotide, is administered inconjunction with a genetic vaccine vector.
 4. The method of claim 1,wherein the library of recombinant polynucleotides is created by aprocess selected from the group consisting of DNA shuffling, error-pronePCR, oligonucleotide-directed mutagenesis, uracil-mediated mutagenesis,and repair-deficient host mutagenesis.
 5. The method of claim 1, whereinthe polynucleotide that has a modulatory effect on an immune response isobtained by: (1) recombining at least first and second forms of anucleic acid that is, or encodes a molecule that is, involved inmodulating an immune response, wherein the first and second forms differfrom each other in two or more nucleotides, to produce a library ofrecombinant polynucleotides; and (2) screening the library to identifyat least one optimized recombinant polynucleotide that exhibits, eitherby itself or through the encoded molecule, an enhanced ability tomodulate an immune response than a form of the nucleic acid from whichthe library was created.
 6. The method of claim 5, wherein the methodfurther comprises the steps of: (3) recombining at least one optimizedrecombinant polynucleotide with a further form of the nucleic acid,which is the same or different from the first and second forms, toproduce a further library of recombinant polynucleotides; (4) screeningthe further library to identify at least one further optimizedrecombinant polynucleotide that exhibits an enhanced ability to modulatean immune response than a form of the nucleic acid from which thelibrary was created; and (5) repeating (3) and (4), as necessary, untilthe further optimized recombinant polynucleotide exhibits an furtherenhanced ability to modulate an immune response than a form of thenucleic acid from which the library was created.
 7. The method of claim1, wherein the optimized recombinant polynucleotide encodes a peptide orpolypeptide that can interact with a cellular receptor involved inmediating an immune response, wherein the peptide or polypeptide acts asan agonist or antagonist of the receptor.
 8. The method of claim 7,wherein the cellular receptor is a macrophage scavenger receptor.
 9. Themethod of claim 7, wherein the cellular receptor is selected from thegroup consisting of a cytokine receptor and a chemokine receptor. 10.The method of claim 9, wherein the chemokine receptor is CCR6.
 11. Themethod of claim 7, wherein the peptide or polypeptide mimics theactivity of a natural ligand for the receptor but does not induce immunereactivity to the natural ligand.
 12. The method of claim 7, wherein thelibrary is screened by: expressing the recombinant polynucleotides sothat the encoded peptides or polypeptides are produced as fusions with aprotein displayed on the surface of a replicable genetic package;contacting the replicable genetic packages with a plurality of cellsthat display the receptor; and identifying cells that exhibit amodulation of an immune response mediated by the receptor.
 13. Themethod of claim 12, wherein the replicable genetic package is selectedfrom the group consisting of a bacteriophage, a cell, a spore, and avirus.
 14. The method of claim 13, wherein the replicable geneticpackage is an Ml 3 bacteriophage and the protein is encoded by geneIIIor geneVII.
 15. The method of claim 7, which method further comprisesintroducing the optimized recombinant polynucleotide into a geneticvaccine vector and administering the vector to a mammal, wherein thepeptide or polypeptide is expressed and acts as an agonist or antagonistof the receptor.
 16. The method of claim 7, which method furthercomprises producing the peptide or polypeptide encoded by the optimizedrecombinant polynucleotide and introducing the peptide or polypeptideinto a mammal in conjunction with a genetic vaccine vector.
 17. Themethod of claim 7, wherein the optimized recombinant polynucleotide isinserted into an antigen-encoding nucleotide sequence of a geneticvaccine vector.
 18. The method of claim 17, wherein the optimizedrecombinant polypeptide is introduced into a nucleotide sequence thatencodes an M-loop of an HBsAg polypeptide.
 19. The method of claim 1,wherein the optimized recombinant polynucleotide comprises a nucleotidesequence rich in unmethylated CpG.
 20. The method of claim 1, whereinthe optimized recombinant polynucleotide encodes a polypeptide thatinhibits an allergic reaction.
 21. The method of claim 20, wherein thepolypeptide is selected from the group consisting of interferon-α,interferon-γ, IL-10, IL-12, an antagonist of IL-4, an antagonist ofIL-5, and an antagonist of IL-13.
 22. The method of 1, wherein theoptimized recombinant polynucleotide encodes an antagonist of IL-10. 23.The method of claim 22, wherein the antagonist of IL-10 is soluble ordefective IL-10 receptor or IL-20/MDA-7.
 24. The method of claim 1,wherein the optimized recombinant polynucleotide encodes a costimulator.25. The method of claim 24, wherein the costimulator is B7-1 (CD80) orB7-2 (CD86) and the screening step involves selecting variants withaltered activity through CD28 or CTLA-4.
 26. The method of claim 24,wherein the costimulator is CD1, CD40, CD154 (ligand for CD40) or CD150(SLAM).
 27. The method of claim 24, wherein the costimulator is acytokine.
 28. The method of claim 27, wherein the cytokine is selectedfrom the group consisting of IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7,IL-8, IL-9, IL-10, IL-11, IL-12, IL-13, IL-14, IL-15, IL-16, IL-17,IL-18, GM-CSF, G-CSF, TNF-α, IFN-α, IFN-γ, and IL-20 (MDA-7).
 29. Themethod of 28, wherein the library of recombinant polynucleotides isscreened by testing the ability of cytokines encoded by the recombinantpolynucleotides to activate cells which contain a receptor for thecytokine.
 30. The method of claim 29, wherein the cells contain aheterologous nucleic acid that encodes the receptor for the cytokine.31. The method of 28, wherein the cytokine is interleukin-12 and thescreening is performed by: growing mammalian cells which contain thegenetic vaccine vector in a culture medium; and detecting whether T cellproliferation or T cell differentiation is induced by contact with theculture medium.
 32. The method of 28, wherein the cytokine isinterferon-α and the screening is performed by: expressing therecombinant polynucleotides so that the encoded peptides or polypeptidesare produced as fusions with a protein displayed on the surface of areplicable genetic package; contacting the replicable genetic packageswith a plurality of B cells; and identifying phage library members thatare capable of inhibiting proliferation of the B cells.
 33. The methodof claim 28, wherein the immune response of interest is differentiationof T cells to T_(H)1 cells and the screening is performed by contactinga population of T cells with the cytokines encoded by the members of thelibrary of recombinant polynucleotides and identifying library membersthat encode a cytokine that induces the T cells to produce IL-2 andinterferon-γ.
 34. The method of claim 27, wherein the cytokine encodedby the optimized recombinant polynucleotide exhibits reducedimmunogenicity compared to a cytokine encoded by a non-optimizedpolynucleotide, and the reduced immunogenicity is detected byintroducing a cytokine encoded by the recombinant polynucleotide into amammal and determining whether an immune response is induced against thecytokine.
 35. The method of claim 24, wherein the costimulator is B7-1(CD80) or B7-2 (CD86) and the cell is tested for ability to costimulatean immune response.
 36. The method of claim 1, wherein the optimizedrecombinant polynucleotide encodes a cytokine antagonist.
 37. The methodof claim 36, wherein the cytokine antagonist is selected from the groupconsisting of a soluble cytokine receptor and a transmembrane cytokinereceptor having a defective signal sequence.
 38. The method of claim 36,wherein the cytokine antagonist is selected from the group consisting ofΔIL-10R and ΔIL-4R.
 39. The method of claim 1, wherein the optimizedrecombinant polynucleotide encodes a polypeptide capable of inducing apredominantly T_(H)1 immune response.
 40. The method of claim 1, whereinthe optimized recombinant polynucleotide encodes a polypeptide capableof inducing a predominantly T_(H)2 immune response.
 41. A method forobtaining a polynucleotide that encodes an accessory molecule thatimproves the transport or presentation of antigens by a cell, the methodcomprising: creating a library of recombinant polynucleotides bysubjecting to recombination nucleic acids that encode all or part of theaccessory molecule; and screening the library to identify an optimizedrecombinant polynucleotide that encodes a recombinant accessory moleculethat confers upon a cell an increased or decreased ability to transportor present an antigen on a surface of the cell compared to an accessorymolecule encoded by the non-recombinant nucleic acids.
 42. The method ofclaim 41, wherein the screening involves: introducing the library ofrecombinant polynucleotides into a genetic vaccine vector that encodesan antigen to form a library of vectors; introducing the library ofvectors into mammalian cells; and identifying mammalian cells thatexhibit increased or decreased immunogenicity to the antigen.
 43. Themethod of claim 41, wherein the accessory molecule comprises aproteasome or a TAP polypeptide.
 44. The method of claim 41, wherein theaccessory molecule comprises a cytotoxic T-cell inducing sequence. 45.The method of claim 44, wherein the cytotoxic T-cell inducing sequenceis obtained from a hepatitis B surface antigen.
 46. The method of claim41, wherein the accessory molecule comprises an immunogenic agonistsequence.